WO2000012764A9 - Techniques permettant d'identifier des sequences de polynucleotides et de polypeptides associees a des etats physiologiques et medicaux - Google Patents

Techniques permettant d'identifier des sequences de polynucleotides et de polypeptides associees a des etats physiologiques et medicaux

Info

Publication number
WO2000012764A9
WO2000012764A9 PCT/US1999/020209 US9920209W WO0012764A9 WO 2000012764 A9 WO2000012764 A9 WO 2000012764A9 US 9920209 W US9920209 W US 9920209W WO 0012764 A9 WO0012764 A9 WO 0012764A9
Authority
WO
WIPO (PCT)
Prior art keywords
human
sequence
sequences
protein
polypeptide
Prior art date
Application number
PCT/US1999/020209
Other languages
English (en)
Other versions
WO2000012764A1 (fr
Inventor
James M Sikela
Walter Messier
Original Assignee
Genoplex Inc
James M Sikela
Walter Messier
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from PCT/US1999/001964 external-priority patent/WO1999039006A2/fr
Application filed by Genoplex Inc, James M Sikela, Walter Messier filed Critical Genoplex Inc
Priority to AU58058/99A priority Critical patent/AU5805899A/en
Publication of WO2000012764A1 publication Critical patent/WO2000012764A1/fr
Publication of WO2000012764A9 publication Critical patent/WO2000012764A9/fr
Priority to JP2001515335A priority patent/JP2004500036A/ja
Priority to AT00960208T priority patent/ATE374834T1/de
Priority to AU71399/00A priority patent/AU784869B2/en
Priority to DK00960208T priority patent/DK1250449T3/da
Priority to PCT/US2000/040517 priority patent/WO2001011088A2/fr
Priority to EP00960208A priority patent/EP1250449B1/fr
Priority to ES00960208T priority patent/ES2292467T3/es
Priority to CA002390094A priority patent/CA2390094A1/fr
Priority to DE60036645T priority patent/DE60036645T2/de

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism

Definitions

  • TECHNICAL FIELD This invention relates to using molecular and evolutionary techniques to identify polynucleotide and polypeptide sequences corresponding to evolved traits that may be relevant to human diseases or conditions, such as unique or enhanced human brain functions, longer human life spans, susceptibility or resistance to development of infectious disease (such as AIDS and hepatitis C), susceptibility or resistance to development of cancer, and aesthetic traits, such as hair growth, susceptibility or resistance to acne, or enhanced muscle mass.
  • infectious disease such as AIDS and hepatitis C
  • susceptibility or resistance to development of cancer and aesthetic traits, such as hair growth, susceptibility or resistance to acne, or enhanced muscle mass.
  • brain function e.g.,
  • K A /K s -type methods includes this and similar methods. These methods have been used to demonstrate the occurrence of Darwinian molecular-level positive selection, resulting in amino acid differences in homologous proteins. Several groups have used such methods to document that a particular protein has evolved more rapidly than the neutral substitution rate, and thus supports the existence of Darwinian molecular-level positive selection. For example, McDonald and Kreitman (1991) Nature 351 :652-654 propose a statistical test of neutral protein evolution hypothesis based on comparison of the number of amino acid replacement substitutions to synonymous substitutions in the coding region of a locus.
  • Evol 13(2):397-406 use the McDonald & Kreitman test as well as a method attributed to Nei and Gojobori, Nei and Jin, and Kumar, Tamura, and Nei; examining the average proportions of P n , the replacement substitutions per replacement site, and P s , the silent substitutions per silent site, to look for evidence of positive selection on bindin genes in sea urchins to investigate whether they have rapidly evolved as a prelude to species formation.
  • Goodwin et al. (1996) Mol. Biol. Evol. 13(2):346-358 uses similar methods to examine the evolution of a particular murine gene family and conclude that the methods provide important fundamental insights into how selection drives genetic divergence in an experimentally manipulatable system.
  • Edwards et al (1995) use degenerate primers to pull out MHC loci from various species of birds and an alligator species, which are then analyzed by the Nei and Gojobori methods (d N : d s ratios) to extend MHC studies to nonmammalian vertebrates.
  • Whitfield et al. (1993) Nature 364:713-715 use Ka/Ks analysis to look for directional selection in the regions flanking a conserved region in the SRy gene (that determines male sex). They suggest that the rapid evolution of SR Y could be a significant cause of reproductive isolation, leading to new species.
  • Wettsetin et al. (1996) Mol. Biol. Evol. 13(l):56-66 apply the MEGA program of Kumar, Tamura and Nei and phylogenetic analysis to investigate the diversification of MHC class I genes in squirrels and related rodents.
  • Parham and Ohta (1996) Science 272:67-74 state that a population biology approach, including tests for selection as well as for gene conversion and neutral drift are required to analyze the generation and maintenance of human MHC class I polymorphism. Hughes (1997) Mol. Biol. Evol.
  • the identification of human genes that have evolved to confer unique or enhanced human functions compared to homologous chimpanzee genes could be applied to developing agents to modulate these unique human functions or to restore function when the gene is defective.
  • the identification of the underlying chimpanzee (or other non-human primate) genes and the specific nucleotide changes that have evolved, and the further characterization of the physical and biochemical changes in the proteins encoded by these evolved genes, could provide valuable information, for example, on what determines susceptibility and resistance to infectious diseases, such as AIDS, what determines susceptibility or resistance to the development of certain cancers, what determines susceptibility or resistance to acne, how hair growth can be controlled, and how to control the formation of muscle versus fat. This valuable information could be applied to developing agents that cause the human proteins to behave more like their chimpanzee homologues.
  • the present invention provides methods for identifying polynucleotide and polypeptide sequences having evolutionarily significant changes, which are associated with physiological conditions, including medical conditions.
  • the invention applies comparative primate genomics to identify specific gene changes which may be associated with, and thus responsible for, physiological conditions, such as medically or commercially relevant evolved traits, and using the information obtained from these evolved genes to develop human treatments.
  • the non-human primate sequences employed in the methods described herein may be any non-human primate, and is preferably a member of the hominoid group, more preferably a chimpanzee, bonobo, gorilla and/or orangutan, and most preferably a chimpanzee.
  • a non-human primate polynucleotide or polypeptide has undergone natural selection that resulted in a positive evolutionarily significant change (i.e., the non-human primate polynucleotide or polypeptide has a positive attribute not present in humans).
  • the positively selected polynucleotide or polypeptide may be associated with susceptibility or resistance to certain diseases or with other commercially relevant traits. Examples of this embodiment include, but are not limited to, polynucleotides and polypeptides that are positively selected in non-human primates, preferably chimpanzees, that may be associated with susceptibility or resistance to infectious diseases and cancer.
  • An example of a commercially relevant trait may include aesthetic traits such as hair growth, muscle mass, susceptibility or resistance to acne.
  • An example of this embodiment includes polynucleotides and polypeptides associated with the susceptibility or resistance to HIV dissemination, propagation and/or development of AIDS.
  • the present invention can thus be useful in gaining insight into the molecular mechanisms that underlie resistance to HIV dissemination, propagation and/or development of AIDS, providing information that can also be useful in discovering and/or designing agents such as drugs that prevent and/or delay development of AIDS.
  • Specific genes that have been positively selected in chimpanzees that may relate to AIDS or other infectious diseases are ICAM-1, ICAM-2, ICAM-3 and MIP-
  • 1- ⁇ . 17- ⁇ -hydroxysteroid dehydrogenase Type IV is a specific gene has been positively selected in chimpanzees that may relate to cancer.
  • a human polynucleotide or polypeptide has undergone natural selection that resulted in a positive evolutionarily significant change (i.e., the human polynucleotide or polypeptide has a positive attribute not present in non-human primates).
  • the polynucleotide or polypeptide may be associated with unique or enhanced functional capabilities of the human brain compared to non-human primates. Another is the longer life-span of humans compared to non-human primates.
  • the present invention can thus be useful in gaining insight into the molecular mechanisms that underlie unique or enhanced human functions, providing information which can also be useful in designing agents such as drugs that modulate such unique or enhanced human functions, and in designing treatment of diseases or conditions related to humans.
  • the present invention can thus be useful in gaining insight into the molecular mechanisms that underlie human cognitive function, providing information which can also be useful in designing agents such as drugs that enhance human brain function, and in designing treatment of diseases related to human brain.
  • a specific example of a human gene that has positive evolutionarily significant changes when compared to non-human primates is a tyrosine kinase gene, KI AA 641.
  • the invention provides methods for identifying a polynucleotide sequence encoding a polypeptide, wherein said polypeptide may be associated with a physiological condition (such as a medically or commercial relevant positive evolutionarily significant change).
  • a physiological condition such as a medically or commercial relevant positive evolutionarily significant change.
  • the positive evolutionarily significant change can be found in humans or in non-human primates.
  • methods are provided for identifying a non- human polynucleotide sequence encoding a polypeptide, wherein said polypeptide may be associated with a physiological condition in the non-human primate, including but not limited to those physiological conditions listed (and throughout the specification), such as susceptibility or resistance to the development of a medically relevant disease state, such as an infectious disease (including viral disease, such as AIDS) or cancer.
  • a physiological condition in the non-human primate including but not limited to those physiological conditions listed (and throughout the specification), such as susceptibility or resistance to the development of a medically relevant disease state, such as an infectious disease (including viral disease, such as AIDS) or cancer.
  • methods comprise the steps of a) comparing non-human primate, preferably a chimpanzee, protein-coding polynucleotide sequences to protein-coding polynucleotide sequences of a human, wherein said human does not have the physiological condition; and b) selecting a non- human polynucleotide sequence that contains a nucleotide change as compared to corresponding sequence of the human, wherein said change is evolutionarily significant.
  • the non-human protein-coding sequences correspond to cDNA.
  • the sequences compared are from brain.
  • the non-human protein coding sequence (and/or the polypeptide encoded therein) may be associated with development and/or maintenance of a physiological trait.
  • the physiological condition may be any physiological condition, including those listed herein, such as, for example, disease (including susceptibility or resistance to disease) such as cancer, infectious disease (including viral diseases such as AIDS); life span; brain function, including cognitive function.
  • disease including susceptibility or resistance to disease
  • infectious disease including viral diseases such as AIDS
  • life span such as cancer, infectious disease (including viral diseases such as AIDS); life span; brain function, including cognitive function.
  • methods for identifying a polynucleotide sequence encoding a human polypeptide, wherein said polypeptide may be associated with a physiological condition that is present in human(s), comprising the steps of: a) comparing human protein-coding polynucleotide sequences to protein-coding polynucleotide sequences of a non-human primate, wherein the non-human primate does not have the physiological condition; and b) selecting a human polynucleotide sequence that contains a nucleotide change as compared to corresponding sequence of the non-human primate, wherein said change is evolutionarily significant.
  • the human protein coding sequence (and/or the polypeptide encoded therein) may be associated with development and/or maintenance of a physiological condition.
  • the human protein-coding sequences correspond to cDNA.
  • the sequences compared are from brain.
  • the physiological condition is life span.
  • the physiological condition is a brain function.
  • the brain function is cognitive function.
  • methods comprise the steps of: (a) comparing human protein-coding nucleotide sequences to protein-coding nucleotide sequences of a non-human primate, preferably a chimpanzee, that is resistant to a particular medically relevant disease state, wherein the human protein coding sequence is associated with development of the disease; and (b) selecting a non- human polynucleotide sequence that contains at least one nucleotide change as compared to the corresponding sequence of the human, wherein the change is evolutionarily significant.
  • the sequences identified by these methods may be further characterized and/or analyzed to confirm that they are associated with the development of the disease state or condition.
  • the most preferred disease states that are applicable to these methods are cancer and infectious diseases, including AIDS, hepatitis C and leprosy.
  • the invention provides methods for identifying a polynucleotide sequence encoding a polypeptide, wherein said polypeptide may be associated with resistance to development of AIDS, comprising the steps of: (a) comparing AIDS resistant non-human primate protein coding sequences to human protein coding sequences, wherein the human protein coding sequences are associated with development of AIDS; and (b) selecting an AIDS resistant non-human primate sequence that contains at least one nucleotide change as compared to the corresponding human sequence, wherein the nucleotide change is evolutionarily significant.
  • these methods can be accomplished, for example, by aligning sequences according to their sequence homology and identifying a human polynucleotide sequence that comprises at least one unique nucleotide change over the corresponding polynucleotide sequence of the non-human primate, wherein the unique nucleotide change is positively selected according to an evolutionary analysis (as described herein).
  • methods for identifying an evolutionarily significant change in a human brain protein-coding polynucleotide sequence, comprising the steps of a) comparing human brain protein-coding polynucleotide sequences to corresponding sequences of a non-human primate; and b) selecting a human polynucleotide sequence that contains a nucleotide change as compared to corresponding sequence of the non-human primate, wherein said change is evolutionarily significant.
  • the human brain protein coding nucleotide sequences correspond to human brain cDNAs.
  • Another aspect of the invention includes methods for identifying a positively selected human evolutionarily significant change. These methods comprise the steps of: (a) comparing human protein-coding nucleotide sequences to protein-coding nucleotide sequences of a non-human primate; and (b) selecting a human polynucleotide sequence that contains at least one (i.e., one or more) nucleotide change as compared to corresponding sequence of the non-human primate, wherein said change is evolutionarily significant.
  • the sequences identified by this method may be further characterized and/or analyzed for their possible association with biologically or medically relevant functions unique or enhanced in humans.
  • Another embodiment of the present invention is a method for large scale sequence comparison between human protein-coding polynucleotide sequences and the protein-coding polynucleotide sequences from a non-human primate, e.g., chimpanzee, comprising: (a) aligning the human polynucleotide sequences with corresponding polynucleotide sequences from non-human primate according to sequence homology; and (b) identifying any nucleotide changes within the human sequences as compared to the homologous sequences from the non-human primate, wherein the changes are evolutionarily significant.
  • the protein coding sequences are from brain.
  • a nucleotide change identified by any of the methods described herein is a non-synonymous substitution.
  • the evolutionary significance of the nucleotide change is determined according to the non- synonymous substitution rate (K A ) of the nucleotide sequence.
  • the evolutionarily significant changes are assessed by determining the K A /K S ratio between the human gene and the homologous gene from non-human primate (such as chimpanzee), and preferably that ratio is at least about 0.75, more preferably greater than about 1 (unity) (i.e., at least about 1), more preferably at least about 1.25, more preferably at least about 1.50, and more preferably at least about 2.00.
  • a positively selected gene has been identified between human and a non-human primate (such as chimpanzee)
  • further comparisons are performed with other non-human primates to confirm whether the human or the non-human primate (such as chimpanzee) gene has undergone positive selection.
  • the invention provides methods for correlating an evolutionarily significant human nucleotide change to a physiological condition in a human (or humans), which comprise analyzing a functional effect (which includes determining the presence of a functional effect), if any, of (the presence or absence of) a polynucleotide sequence identified by any of the methods described herein, wherein presence of a functional effect indicates a correlation between the evolutionarily significant nucleotide change and the physiological condition.
  • a functional effect if any may be assessed using a polypeptide sequence (or a portion of the polypeptide sequence) encoded by a nucleotide sequence identified by any of the methods described herein.
  • the present invention also provides comparison of the identified polypeptides by physical and biochemical methods widely used in the art to determine the structural or biochemical consequences of the evolutionarily significant changes.
  • Physical methods are meant to include methods that are used to examine structural changes to proteins encoded by genes found to have undergone adaptive evolution.
  • the invention provides methods for identifying a target site (which includes one or more target sites) which may be suitable for therapeutic intervention, comprising comparing a human polypeptide (or a portion of the polypeptide) encoded in a sequence identified by any of the methods described herein, with a corresponding non-human polypeptide (or a portion of the polypeptide), wherein a location of a molecular difference, if any, indicates a target site.
  • the invention provides methods for identifying a target site (which includes one or more target sites) which may be suitable for therapeutic intervention, comprising comparing a non-human polypeptide (or a portion of the polypeptide) encoded in a sequence identified by any of the methods described herein, with a corresponding human polypeptide (or a portion of the polypeptide), wherein a location of a molecular difference, such as an amino acid difference, if any, indicates a target site.
  • Biochemical methods are meant to include methods that are used to examine functional differences, such as binding specificity, binding strength, or optimal binding conditions, for a protein encoded by a gene that has undergone adaptive evolution. Side-by-side comparison of biochemical characteristics of a protein (either human or non-human primate) and the evolved homologous protein (either non- human primate or human, respectively) will reveal valuable information for developing treatments for related human conditions and diseases.
  • the invention provides methods of identifying an agent which may modulate a physiological condition, said method comprising contacting an agent (i.e., at least one agent to be tested) with a cell that has been transfected with a polynucleotide sequence identified by any of the methods described herein, wherein an agent is identified by its ability to modulate function of the polynucleotide sequence.
  • the invention provides methods of identifying an agent which may modulate a physiological condition, said method comprising contacting an agent (i.e., at least one agent) to be tested with a polypeptide (or a fragment of a polypeptide and/or a composition comprising a polypeptide or fragment of a polypeptide) encoded in or within a polynucleotide identified by any of the methods described herein, wherein an agent is identified by its ability to modulate function of the polypeptide.
  • an agent i.e., at least one agent
  • a polypeptide or a fragment of a polypeptide and/or a composition comprising a polypeptide or fragment of a polypeptide
  • the invention also provides agents which are identified using the screening methods described herein.
  • the invention provides methods of screening agents which may modulate the activity of the human polynucleotide or polypeptide to either modulate a unique or enhanced human function or to mimic the non-human primate trait of interest, such as susceptibility or resistance to development of a disease, such as AIDS.
  • These methods comprise contacting a cell which has been transfected with a polynucleotide sequence with an agent to be tested, and identifying agents based on their ability to modulate function of the polynucleotide or contacting a polypeptide preparation with an agent to be tested and identifying agents based upon their ability to modulate function of the polypeptide.
  • Figure 1 depicts a phylogenetic tree for primates within the hominoid group.
  • the branching orders are based on well-supported mitochondrial DNA phylogenies. Messier and Stewart (1997) Nature 385:151-154.
  • Figure 2 is a nucleotide sequence alignment between human and chimpanzee ICAM-1 sequences (GenBank accession numbers X06990 and X86848, respectively). The amino acid translation of the chimpanzee sequence is shown below the alignment.
  • Figure 3 shows the nucleotide sequence of gorilla ICAM-1 (SEQ ID NO:4).
  • Figure 4 shows the nucleotide sequence of orangutan ICAM-1 (SEQ ID NO:5).
  • Figures 5(A)-(E) show the polypeptide sequence alignment of ICAM-1 from several primate species.
  • Figures 6(A)-(B) show the polypeptide sequence alignment of ICAM-2 from several primate species.
  • Figures7(A)-(P) show the polypeptide sequence alignment of ICAM-3 from several primate species.
  • Figure 8 depicts a schematic representation of a procedure for comparing human/primate brain polynucleotides, selecting sequences with evolutionarily significant changes, and further characterizing the selected sequences.
  • the diagram of Figure 8 illustrates a preferred embodiment of the invention and together with the description serves to explain the principles of the invention, along with elaboration and optional additional steps. It is understood that any human/primate polynucleotide sequence can be compared by a similar procedure and that the procedure is not limited to brain polynucleotides.
  • the present invention applies comparative genomics to identify specific gene changes which are associated with, and thus may contribute to or be responsible for, physiological conditions, such as medically or commercially relevant evolved traits.
  • the invention comprises a comparative genomics approach to identify specific gene changes responsible for differences in functions and diseases distinguishing humans from other non-humans, particularly primates, and most preferably chimpanzees, including the two known species, common chimpanzees and bonobos (pygmy chimpanzees).
  • chimpanzees and humans are 98.5% identical at the DNA sequence level and the present invention can identify the adaptive molecular changes underlying differences between the species in a number of areas, including unique or enhanced human cognitive abilities and chimpanzee resistance to AIDS and certain cancers.
  • the present invention provides exact information on evolutionary solutions that eliminate disease or provide unique functions.
  • the present invention identifies genes that have evolved to confer an evolutionary advantage and the specific evolved changes.
  • human protein-coding polynucleotides may contain sequence changes that are found in humans but not in other evolutionarily closely related species such as non-human primates, as a result of adaptive selection during evolution.
  • the present invention further results from the observation that the genetic information of non-human primates may contain changes that are found in a particular non-human primate but not in humans, as a result of adaptive selection during evolution.
  • a non-human primate polynucleotide or polypeptide has undergone natural selection that resulted in a positive evolutionarily significant change (i.e., the non-human primate polynucleotide or polypeptide has a positive attribute not present in humans).
  • the positively selected polynucleotide or polypeptide may be associated with susceptibility or resistance to certain diseases or other commercially relevant traits.
  • Medically relevant examples of this embodiment include, but are not limited to, polynucleotides and polypeptides that are positively selected in non-human primates, preferably chimpanzees, that may be associated with susceptibility or resistance to infectious diseases and cancer.
  • An example of this embodiment includes polynucleotides and polypeptides associated with the susceptibility or resistance to progression from HIV infection to development of AIDS.
  • the present invention can thus be useful in gaining insight into the molecular mechanisms that underlie resistance to progression from HIV infection to development of AIDS, providing information that can also be useful in discovering and/or designing agents such as drugs that prevent and/or delay development of AIDS.
  • polynucleotides and polypeptides that are positively selected in non-human primates that may be associated with aesthetic traits, such as hair growth, acne or muscle mass.
  • Positively selected human evolutionarily significant changes in polynucleotide and polypeptide sequences may be attributed to human capabilities that provide humans with competitive advantages, particularly when compared to the closest evolutionary relative, chimpanzee, such as unique or enhanced human brain functions.
  • the present invention identifies human genes that evolved to provide unique or enhanced human cognitive abilities and the actual protein changes that confer functional differences will be quite useful in therapeutic approaches to treat cognitive deficiencies as well as cognitive enhancement for the general population.
  • a "polynucleotide” refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides, or analogs thereof. This term refers to the primary structure of the molecule, and thus includes double- and single-stranded DNA, as well as double- and single-stranded RNA. It also includes modified polynucleotides such as methylated and/or capped polynucleotides. The terms "polynucleotide” and “nucleotide sequence” are used interchangeably.
  • a “gene” refers to a polynucleotide or portion of a polynucleotide comprising a sequence that encodes a protein. It is well understood in the art that a gene also comprises non-coding sequences, such as 5' and 3' flanking sequences (such as promoters, enhancers, repressors, and other regulatory sequences) as well as introns.
  • polypeptide polypeptide
  • peptide protein
  • proteins are used interchangeably herein to refer to polymers of amino acids of any length. These terms also include proteins that are post-translationally modified through reactions that include glycosylation, acetylation and phosphorylation.
  • a “physiological condition” is a term well-understood in the art and means any condition or state that be measured and/or observed.
  • a “physiological condition” includes, but is not limited to, a physical condition, such as degree of body fat, alopecia (baldness), acne; life-expectancy; disease states (which include susceptibility and/or resistance to diseases), such as cancer or infectious diseases. Examples of physiological conditions are provided below (see, e.g., definitions of "human medically relevant medical condition”, “human commercially relevant condition”, “medically relevant evolved trait”, and “commercially relevant evolved trait”) and throughout the specification, and it is understood that these terms and examples refer to a physiological condition.
  • a physiological condition may be, but is not necessarily, the result of multiple factors, any of which in turn may be considered a physiological condition.
  • a physiological condition which is "present" in a human or non-human primate occurs within a given population, and includes those physiological conditions which are unique and/or enhanced in a given population when compared to another population.
  • human medically relevant condition or “human commercially relevant condition” are used herein to refer to human conditions for which medical or non-medical (respectively) intervention is desired.
  • medically relevant evolved trait is used herein to refer to traits that have evolved in humans or non-human primates whose analysis could provide information (e.g., physical or biochemical data) relevant to the development of a human medical treatment.
  • commercially relevant evolved trait is used herein to refer to traits that have evolved in humans or non-human primates whose analysis could provide information (e.g., physical or biochemical data) relevant to the development of a non- medical product or treatment for human use.
  • K A /K s -type methods means methods that evaluate differences, frequently (but not always) shown as a ratio, between the number of nonsynonymous substitutions and synonymous substitutions in homologous genes (including the more rigorous methods that determine non-synonymous and synonymous sites). These methods are designated using several systems of nomenclature, including but not limited to K A /K S , d N /d s , D N /D S .
  • evolutionarily significant change or “adaptive evolutionary change” refers to one or more nucleotide or peptide sequence change(s) between two species that may be attributed to a positive selective pressure.
  • One method for determining the presence of an evolutionarily significant change is to apply a K A /K S - type analytical method, such as to measure a K A /K S ratio.
  • a K A /K S ratio 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 most preferably at least about 2.0 indicates the action of positive selection and is considered to be an evolutionarily significant change.
  • positive evolutionarily significant change means an evolutionarily significant change in a particular species that results in an adaptive change that is positive as compared to other related species.
  • positive evolutionarily significant changes are changes that have resulted in enhanced cognitive abilities in humans and adaptive changes in chimpanzees that have resulted in the ability of the chimpanzees infected with HIV to be resistant to progression to full-blown AIDS.
  • resistant means that an organism, such as a chimpanzee, exhibits an ability to avoid, or diminish the extent of, a disease condition and/or development of the disease, preferably when compared to non-resistant organisms, typically humans.
  • a chimpanzee is resistant to certain impacts of HIV and other viral infections, and/or it does not develop the ultimate disease - AIDS.
  • susceptibility means that an organism, such as a human, fails to avoid, or diminish the extent of, a disease condition and/or development of the disease condition, preferably when compared to an organism that is known to be resistant, such as a non-human primate, such as chimpanzee.
  • a human is susceptible to certain impacts of HIV and other viral infections and/or development of the ultimate disease - AIDS.
  • resistance .and susceptibility vary from individual to individual, and that, for purposes of this invention, these terms also apply to a group of individuals within a species, and comparisons of resistance and susceptibility generally refer to overall, average differences between species, although intra-specific comparisons may be used.
  • homologous sequences are compared.
  • homologous sequences or “homologues” or “orthologs” are thought, believed, or known to be functionally related.
  • a functional relationship may be indicated in any one of a number of ways, including, but not limited to, (a) degree of sequence identity; (b) same or similar biological function. Preferably, both (a) and (b) are indicated.
  • the degree of sequence identity may vary, but is preferably at least 50%o (when using standard sequence alignment programs known in the art), more preferably at least 60%), more preferably at least about 75%, more preferably at least about 85%o.
  • Homology can be determined using software programs readily available in the art, such as those discussed in Current Protocols in Molecular Biology (F.M. Ausubel et al, eds., 1987) Supplement 30, section 7.718, Table 7.71.
  • Preferred alignment programs are MacVector (Oxford Molecular Ltd, Oxford, U.K.) and ALIGN Plus (Scientific and Educational Software, Pennsylvania).
  • Another preferred alignment program is Sequencher (Gene Codes, Ann Arbor, Michigan), using default parameters.
  • nucleotide change refers to nucleotide substitution, deletion, and/or insertion, as is well understood in the art.
  • human protein-coding nucleotide sequence which is "associated with susceptibility to AIDS” as used herein refers to a human nucleotide sequence that encodes a protein that is associated with HIV dissemination (within the organism, i.e., intra-organism infectivity), propagation and/or development of AIDS. Due to the extensive research in the mechanisms underlying progression from HIV infection to the development of AIDS, a number of candidate human genes are believed or known to be associated with one or more of these phenomena.
  • a polynucleotide (including any polypeptide encoded therein) sequence associated with susceptibility to AIDS is one which is either known or implicated to play a role in HIV dissemination, replication, and/or subsequent progression to full-blown AIDS. Examples of such candidate genes are provided below.
  • AIDS resistant means that an organism, such as a chimpanzee, exhibits an ability to avoid, or diminish the extent of, the result of HIV infection (such as propagation and dissemination) and/or development of AIDS, preferably when compared to AIDS-susceptible humans.
  • “Susceptibility” to AIDS means that an organism, such as a human, fails to avoid, or diminish the extent of, the result of HIV infection (such as propagation and dissemination) and/or development of AIDS, preferably when compared to an organism that is known to be AIDS resistant, such as a non-human primate, such as chimpanzee.
  • brain protein-coding nucleotide sequence refers to a nucleotide sequence expressed in the brain that encodes a protein.
  • One example of the "brain protein-coding nucleotide sequence” is a brain cDNA sequence.
  • brain functions unique or enhanced in humans or “unique functional capabilities of the human brain” or “brain functional capability that is unique or enhanced in humans” refers to any brain function, either in kind or in degree, that is identified and/or observed to be enhanced in humans compared to other non-human primates.
  • Such brain functions include, but are not limited to high capacity information processing, storage and retrieval capabilities, creativity, memory, language abilities, brain-mediated emotional response, locomotion, pain/pleasure sensation, olfaction, and temperament.
  • “Housekeeping genes” is a term well understood in the art and means those genes associated with general cell function, including but not limited to growth, division, stasis, metabolism, and/or death. "Housekeeping" genes generally perform functions found in more than one cell type. In contrast, cell-specific genes generally perform functions in a particular cell type (such as neurons) and/or class (such as neural cells).
  • the term "agent”, as used herein, means a biological or chemical compound such as a simple or complex organic or inorganic molecule, a peptide, a protein or an oligonucleotide.
  • oligomers such as oligopeptides and oligonucleotides
  • synthetic organic and inorganic compounds based on various core structures, and these are also included in the term "agent”.
  • various natural sources can provide compounds for screening, such as plant or animal extracts, and the like. Compounds can be tested singly or in combination with one another.
  • to modulate function of a polynucleotide or a polypeptide means that the function of the polynucleotide or polypeptide is altered when compared to not adding an agent. Modulation may occur on any level that affects function.
  • a polynucleotide or polypeptide function may be direct or indirect, and measured directly or indirectly.
  • a "function of a polynucleotide” includes, but is not limited to, replication; translation; expression pattern(s).
  • a polynucleotide function also includes functions associated with a polypeptide encoded within the polynucleotide. For example, an agent which acts on a polynucleotide and affects protein expression, conformation, folding (or other physical characteristics), binding to other moieties (such as ligands), activity (or other functional characteristics), regulation and/or other aspects of protein structure or function is considered to have modulated polynucleotide function.
  • a "function of a polypeptide” includes, but is not limited to, conformation, folding (or other physical characteristics), binding to other moieties (such as ligands), activity (or other functional characteristics), and/or other aspects of protein structure or functions.
  • an agent that acts on a polypeptide and affects its conformation, folding (or other physical characteristics), binding to other moieties includes, but is not limited to, conformation, folding (or other physical characteristics), binding to other moieties (such as ligands), activity (or other functional characteristics), and/or other aspects of protein structure or functions.
  • an agent that acts on a polypeptide and affects its conformation, folding (or other physical characteristics), binding to other moieties include, but is not limited to, conformation, folding (or other physical characteristics), binding to other moieties
  • an effective agent can act to modulate the function of a polypeptide include, but are not limited to 1) changing the conformation, folding or other physical characteristics; 2) changing the binding strength to its natural ligand or changing the specificity of binding to ligands; and 3) altering the activity of the polypeptide.
  • modulate susceptibility to development of AIDS and “modulate resistance to development of AIDS”, as used herein, include modulating intra- organism cell-to-cell transmission or infectivity of HIV.
  • the terms further include reducing susceptibility to development of AIDS and/or cell-to-cell transmission or infectivity of HIV.
  • the terms further include increasing resistance to development of AIDS and/or cell-to-cell transmission or infectivity of HIV.
  • One means of assessing whether an agent is one that modulates susceptibility or resistance to development of AIDS is to determine whether at least one index of HIV susceptibility is affected, using a cell-based system as described herein, as compared with an appropriate control.
  • Indicia of HIV susceptibility include, but are not limited to, cell-to-cell transmission of the virus, as measured by total number of cells infected with HIV and syncytia formation.
  • target site means a location in a polypeptide which can beg a single amino acid and/or is a part of, a structural and/or functional motif, e.g., a binding site, a dimerization domain, or a catalytic active site.
  • Target sites may be a useful for direct or indirect interaction with an agent, such as a therapeutic agent.
  • molecular difference includes any structural and/or functional difference. Methods to detect such differences, as well as examples of such differences, are described herein.
  • a "functional effect” is a term well known in the art, and means any effect which is exhibited on any level of activity, whether direct or indirect.
  • the source of the human and non-human polynucleotide can be any suitable source, e.g., genomic sequences or cDNA sequences.
  • cDNA sequences from human and a non-human primate are compared.
  • Human protein-coding sequences can be obtained from public databases such as the Genome Sequence Data Bank and GenBank. These databases serve as repositories of the molecular sequence data generated by ongoing research efforts.
  • human protein-coding sequences may be obtained from, for example, sequencing of cDNA reverse transcribed from mRNA expressed in human cells, or after PCR amplification, according to methods well known in the art.
  • human genomic sequences may be used for sequence comparison.
  • Human genomic sequences can be obtained from public databases or from a sequencing of commercially available human genomic DNA libraries or from genomic DNA, after PCR.
  • the non-human primate protein-coding sequences can be obtained by, for example, sequencing cDNA clones that are randomly selected from a non-human primate cDNA library.
  • the non-human primate cDNA library can be constructed from total mRNA expressed in a primate cell using standard techniques in the art.
  • the cDNA is prepared from mRNA obtained from a tissue at a determined developmental stage, or a tissue obtained after the primate has been subjected to certain environmental conditions.
  • cDNA libraries used for the sequence comparison of the present invention can be constructed using conventional cDNA library construction techniques that are explained fully in the literature of the art.
  • Total mRNAs are used as templates to reverse-transcribe cDNAs. Transcribed cDNAs are subcloned into appropriate vectors to establish a cDNA library.
  • the established cDNA library can be maximized for full-length cDNA contents, although less than full-length cDNAs may be used.
  • the sequence frequency can be normalized according to, for example, Bonaldo et al. (1996) Genome Research 6:791-806.
  • cDNA clones randomly selected from the constructed cDNA library can be sequenced using standard automated sequencing techniques. Preferably, full- length cDNA clones are used for sequencing. Either the entire or a large portion of cDNA clones from a cDNA library may be sequenced, although it is also possible to practice some embodiments of the invention by sequencing as little as a single cDNA, or several cDN A clones.
  • non-human primate cDNA clones to be sequenced can be pre-selected according to their expression specificity.
  • the cDNAs can be subject to subtraction hybridization using mRNAs obtained from other organs, tissues or cells of the same animal. Under certain hybridization conditions with appropriate stringency and concentration, those cDNAs that hybridize with non-tissue specific mRNAs and thus likely represent "housekeeping" genes will be excluded from the cDNA pool. Accordingly, remaining cDNAs to be sequenced are more likely to be associated with tissue-specific functions.
  • non-tissue-specific mRNAs can be obtained from one organ, or preferably from a combination of different organs and cells. The amount of non-tissue-specific mRNAs are maximized to saturate the tissue-specific cDNAs.
  • information from online public databases can be used to select or give priority to cDNAs that are more likely to be associated with specific functions.
  • the non-human primate cDNA candidates for sequencing can be selected by PCR using primers designed from candidate human cDNA sequence.
  • Candidate human cDNA sequences are, for example, those that are only found in a specific tissue, such as brain, or that correspond to genes likely to be important in the specific function, such as brain function.
  • Such human tissue-specific cDNA sequences can be obtained by searching online human sequence databases such as GenBank, in which information with respect to the expression profile and/or biological activity for cDNA sequences are specified.
  • sequences of non-human primate for example, from an AIDS-resistant non- human primate
  • a known human gene may be obtained using methods standard in the art, such as from public databases such as GenBank or PCR methods (using, for example, GeneAmp PCR System 9700 thermocyclers (Applied Biosystems, Inc.)).
  • non-human primate cDNA candidates for sequencing can be selected by PCR using primers designed from candidate human cDNA sequences.
  • primers may be made from the human sequences using standard methods in the art, including publicly available primer design programs such as PRIMER® (Whitehead Institute).
  • the sequence amplified may then be sequenced using standard methods and equipment in the art, such as automated sequencers
  • nucleotide sequences are obtained from a human source and a non-human source.
  • the human and non-human nucleotide sequences are compared to one another to identify sequences that are homologous.
  • the homologous sequences are analyzed to identify those that have nucleic acid sequence differences between the two species.
  • molecular evolution analysis is conducted to evaluate quantitatively and qualitatively the evolutionary significance of the differences. For genes that have been positively selected between two species, e.g., human and chimp, it is useful to determine whether the difference occurs in other non-human primates.
  • the sequence is characterized in terms of molecular/genetic identity and biological function.
  • the information can be used to identify agents useful in diagnosis and treatment of human medically or commercially relevant conditions.
  • the general methods of the invention entail comparing human protein-coding nucleotide sequences to protein-coding nucleotide sequences of a non-human, preferably a primate, and most preferably a chimpanzee.
  • a non-human preferably a primate
  • a chimpanzee examples of other non- human primates are bonobo, gorilla, orangutan, gibbon, Old World monkeys, and
  • FIG. 1 A phylogenetic tree for primates within the hominoid group is depicted in FIG. 1.
  • Bioinformatics is applied to the comparison and sequences are selected that contain a nucleotide change or changes that is/are evolutionarily significant change(s).
  • the invention enables the identification of genes that have evolved to confer some evolutionary advantage and the identification of the specific evolved changes.
  • Protein-coding sequences of human and another non-human primate are compared to identify homologous sequences. Any appropriate mechanism for completing this comparison is contemplated by this invention. Alignment may be performed manually or by software (examples of suitable alignment programs are known in the art).
  • protein-coding sequences from a non-human primate are compared to human sequences via database searches, e.g., BLAST searches.
  • the high scoring "hits," i.e., sequences that show a significant similarity after BLAST analysis, will be retrieved and analyzed. Sequences showing a significant similarity can be those having at least about 60%>, at least about 75%, at least about 80%>, at least about 85%), or at least about 90% sequence identity.
  • sequences showing greater than about 80% identity are further analyzed.
  • the homologous sequences identified via database searching can be aligned in their entirety using sequence alignment methods and programs that are known and available in the art, such as the commonly used simple alignment program CLUSTAL V by Higgins et al. (1992) CABIOS 8:189-191.
  • sequencing and homologous comparison of protein-coding sequences between human and a non-human primate may be performed simultaneously by using the newly developed sequencing chip technology. See, for example, Rava et al. US Patent 5,545,531.
  • the aligned protein-coding sequences of human and another non-human primate are analyzed to identify nucleotide sequence differences at particular sites. Again, any suitable method for achieving this analysis is contemplated by this invention. If there are no nucleotide sequence differences, the non-human primate protein coding sequence is not usually further analyzed.
  • the detected sequence changes are generally, and preferably, initially checked for accuracy. Preferably, the initial checking comprises performing one or more of the following steps, any and all of which are known in the art: (a) finding the points where there are changes between the non-human primate and human sequences; (b) checking the sequence fluorogram
  • nucleotide change encompasses at least one nucleotide change, either a substitution, a deletion or an insertion, in a human protein-coding polynucleotide sequence as compared to corresponding sequence from a non-human primate.
  • the change is a nucleotide substitution. More preferably, more than one substitution is present in the identified human sequence and is subjected to molecular evolution analysis.
  • K A /K s -type methods can be employed to evaluate quantitatively and qualitatively the evolutionary significance of the identified nucleotide changes between human gene sequences and that of a non-human primate.
  • K A nonsynonymous nucleotide substitutions per nonsynonymous site
  • K s synonymous substitutions per synonymous site
  • the K A /K S analysis by Li et al. is used to carry out the present invention, although other analysis programs that can detect positively selected genes between species can also be used.
  • the K A /K S method which comprises a comparison of the rate of nonsynonymous substitutions per non-synonymous site with the rate of synonymous substitutions per synonymous site between homologous protein-coding region of genes in terms of a ratio, is used to identify sequence substitutions that may be driven by adaptive selections as opposed to neutral selections during evolution.
  • a synonymous (“silent") substitution is one that, owing to the degeneracy of the genetic code, makes no change to the amino acid sequence encoded; a non-synonymous substitution results in an amino acid replacement.
  • the extent of each type of change can be estimated as K A and K s , respectively, the numbers of synonymous substitutions per synonymous site and non-synonymous substitutions per non-synonymous site.
  • Calculations of K A /K S may be performed manually or by using software.
  • An example of a suitable program is MEGA (Molecular Genetics Institute, Pennsylvania State University).
  • MEGA Molecular Genetics Institute, Pennsylvania State University.
  • the length of the polynucleotide sequence analyzed can be any appropriate length.
  • the entire coding sequence is compared, in order to determine any and all significant changes.
  • Solid Mol. Evol. 36:96-99 or INA can be used to calculate the K A and K s values for all pairwise comparisons.
  • This analysis can be further adapted to examine sequences in a "sliding window” fashion such that small numbers of important changes are not masked by the whole sequence.
  • “Sliding window” refers to examination of consecutive, overlapping subsections of the gene (the subsections can be of any length).
  • K A /K s The comparison of non-synonymous and synonymous substitution rates is represented by the K A /K s ratio.
  • K A /K s has been shown to be a reflection of the degree to which adaptive evolution has been at work in the sequence under study. Full length or partial segments of a coding sequence can be used for the K A /K. analysis. The higher the K A /K. ratio, the more likely that a sequence has undergone adaptive evolution and the non-synonymous substitutions are evolutionarily significant. See, for example, Messier and Stewart (1997).
  • the K A /K S 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 1.50, or more preferably at least about 2.00.
  • K A /K S ratios significantly greater than unity strongly suggest that positive selection has fixed greater numbers of amino acid replacements than can be expected as a result of chance alone, and is in contrast to the commonly observed pattern in which the ratio is less than or equal to one.
  • Ratios less than one generally signify the role of negative, or purifying selection: there is strong pressure on the primary structure of functional, effective proteins to remain unchanged.
  • All methods for calculating K A /K S ratios are based on a pairwise comparison of the number of nonsynonymous substitutions per nonsynonymous site to the number of synonymous substitutions per synonymous site for the protein-coding regions of homologous genes from related species.
  • Each method implements different corrections for estimating "multiple hits" (i.e., more than one nucleotide substitution at the same site).
  • Each method also uses different models for how DNA sequences change over evolutionary time.
  • a combination of results from different algorithms is used to increase the level of sensitivity for detection of positively-selected genes and confidence in the result.
  • K A /K S ratios should be calculated for orthologous gene pairs, as opposed to paralogous gene pairs (i.e., a gene which results from speciation, as opposed to a gene that is the result of gene duplication) Messier and Stewart (1997).
  • This distinction may be made by performing additional comparisons with other non- human primates, such as gorilla and orangutan, which allows for phylogenetic tree- building.
  • Orthologous genes when used in tree-building will yield the known "species tree", i.e., will produce a tree that recovers the known biological tree.
  • paralogous genes will yield trees which will violate the known biological tree.
  • sequences that are functionally related to human protein-coding sequences.
  • sequences may include, but are not limited to, non-coding sequences or coding sequences that do not encode human proteins.
  • These related sequences can be, for example, physically adjacent to the human protein- coding sequences in the human genome, such as introns or 5'- and 3'- flanking sequences (including control elements such as promoters and enhancers).
  • These related sequences may be obtained via searching a public human genome database such as GenBank or, alternatively, by screening and sequencing a human genomic library with a protein-coding sequence as probe.
  • the evolutionarily significant nucleotide changes which are detected by molecular evolution analysis such as the K A /K S analysis, can be further assessed for their unique occurrence in humans (or the non-human primate) or the extent to which these changes are unique in humans (or the non-human primate). For example, the identified changes can be tested for presence/absence in other non-human primate sequences.
  • sequences with at least one evolutionarily significant change between human and one non-human primate can be used as primers for PCR analysis of other non-human primate protein-coding sequences, and resulting polynucleotides are sequenced to see whether the same change is present in other non-human primates.
  • These comparisons allow further discrimination as to whether the adaptive evolutionary changes are unique to the human lineage as compared to other non- human primates or whether the adaptive change is unique to the non-human primates (i.e., chimpanzee) as compared to humans and other non-human primates.
  • a nucleotide change that is detected in human but not other primates more likely represents a human adaptive evolutionary change.
  • a nucleotide change that is detected in a non-human primate i.e., chimpanzee
  • a non-human primate i.e., chimpanzee
  • Other non-human primates used for comparison can be selected based on their phylogenetic relationships with human. Closely related primates can be those within the hominoid sublineage, such as chimpanzee, bonobo, gorilla, and orangutan. Non-human primates can also be those that are outside the hominoid group and thus not so closely related to human, such as the Old World monkeys and New World monkeys.
  • Sequences with significant changes can be used as probes in genomes from different human populations to see whether the sequence changes are shared by more than one human population.
  • Gene sequences from different human populations can be obtained from databases made available by, for example, the Human Genome
  • Sequences with significant changes between species can be further characterized in terms of their molecular/genetic identities and biological functions, using methods and techniques known to those of ordinary skill in the art.
  • the sequences can be located genetically and physically within the human genome using publicly available bio-informatics programs.
  • the newly identified significant changes within the nucleotide sequence may suggest a potential role of the gene in human evolution and a potential association with human-unique functional capabilities.
  • the putative gene with the identified sequences may be further characterized by, for example, homologue searching. Shared homology of the putative gene with a known gene may indicate a similar biological role or function.
  • Another exemplary method of characterizing a putative gene sequence is on the basis of known sequence motifs. Certain sequence patterns are known to code for regions of proteins having specific biological characteristics such as signal sequences, DNA binding domains, or transmembrane domains.
  • the identified human sequences with significant changes can also be further evaluated by looking at where the gene is expressed in terms of tissue- or cell type- specificity.
  • the identified coding sequences can be used as probes to perform in situ mRNA hybridization that will reveal the expression patterns of the sequences.
  • Genes that are expressed in certain tissues may be better candidates as being associated with important human functions associated with that tissue, for example brain tissue.
  • the timing of the gene expression during each stage of human development can also be determined.
  • the functional roles of the identified nucleotide sequences with significant changes can be assessed by conducting functional assays for different alleles of an identified gene in a model system, such as yeast, nematode, Drosophila, and mouse.
  • Model systems may be cell-based or in vivo, such as transgenic animals.
  • the transgenic mouse system is used. Methods of making cell-based systems and/or transgenic animal systems are known in the art and need not be described in detail herein.
  • chimpanzee ICAM-3 contains a glutamine residue (Q101) at the site in which human ICAM-3 contains a proline (PI 01).
  • the human protein is known to bend sharply at this point. Replacement of the proline by glutamine in the chimpanzee protein is likely to result in a much less sharp bend at this point. This has clear implications for packaging of the ICAM-3 chimpanzee protein into HIN virions.
  • the present invention provides methods for identifying agents that are useful in modulating human-unique or human-enhanced functional capabilities and/or correcting defects in these capabilities using these sequences. These methods employ, for example, screening techniques known in the art, such as in vitro systems, cell-based expression systems and transgenic animal systems.
  • screening techniques known in the art, such as in vitro systems, cell-based expression systems and transgenic animal systems.
  • the approach provided by the present invention not only identifies rapidly evolved genes, but indicates modulations that can be made to the protein that may not be too toxic because they exist in another species.
  • Screening methods also provides screening methods using the polynucleotides and polypeptides identified and characterized using the above- described methods. These screening methods are useful for identifying agents which may modulate the function(s) of the polynucleotides or polypeptides in a manner that would be useful for a human treatment.
  • the methods entail contacting at least one agent to be tested with either a cell that has been transfected with a polynucleotide sequence identified by the methods described above, or a preparation of the polypeptide encoded by such polynucleotide sequence, wherein an agent is identified by its ability to modulate function of either the polynucleotide sequence or the polypeptide.
  • the term "agent” means a biological or chemical compound such as a simple or complex organic or inorganic molecule, a peptide, a protein or an oligonucleotide.
  • a vast array of compounds can be synthesized, for example oligomers, such as oligopeptides and oligonucleotides, and synthetic organic and inorganic compounds based on various core structures, and these are also included in the term "agent".
  • various natural sources can provide compounds for screening, such as plant or animal extracts, and the like. Compounds can be tested singly or in combination with one another.
  • modulate function of a polynucleotide or a polypeptide means that the function of the polynucleotide or polypeptide is altered when compared to not adding an agent. Modulation may occur on any level that affects function.
  • a polynucleotide or polypeptide function may be direct or indirect, and measured directly or indirectly.
  • a "function" of a polynucleotide includes, but is not limited to, replication, translation, and expression pattern(s).
  • a polynucleotide function also includes functions associated with a polypeptide encoded within the polynucleotide.
  • an agent which acts on a polynucleotide and affects protein expression, conformation, folding (or other physical characteristics), binding to other moieties (such as ligands), activity (or other functional characteristics), regulation and/or other aspects of protein structure or function is considered to have modulated polynucleotide function.
  • a "function" of a polypeptide includes, but is not limited to, conformation, folding (or other physical characteristics), binding to other moieties (such as ligands), activity (or other functional characteristics), and/or other aspects of protein structure or functions.
  • an agent that acts on a polypeptide and affects its conformation, folding (or other physical characteristics), binding to other moieties (such as ligands), activity (or other functional characteristics), and/or other aspects of protein structure or functions is considered to have modulated polypeptide function.
  • the choice of agents to be screened is governed by several parameters, such as the particular polynucleotide or polypeptide target, its perceived function, its three-dimensional structure (if known or surmised), and other aspects of rational drug design. Techniques of combinatorial chemistry can also be used to generate numerous permutations of candidates. Those of skill in the art can devise and/or obtain suitable agents for testing.
  • an in vivo screening assay may have several advantages over conventional drug screening assays: 1) if an agent must enter a cell to achieve a desired therapeutic effect, an in vivo assay can give an indication as to whether the agent can enter a cell; 2) an in vivo screening assay can identify agents that, in the state in which they are added to the assay system are ineffective to elicit at least one characteristic which is associated with modulation polynucleotide or polypeptide function, but that are modified by cellular components once inside a cell in such a way that they become effective agents; 3) most importantly, an in vivo assay system allows identification of agents affecting any component of a pathway that ultimately results in characteristics that are associated with polynucleotide or polypeptide function.
  • screening can be performed by adding an agent to a sample of appropriate cells which have been transfected with a polynucleotide identified using the methods of the present invention, and monitoring the effect, i.e., modulation of a function of the polynucleotide or the polypeptide encoded within the polynucleotide.
  • the experiment preferably includes a control sample which does not receive the candidate agent.
  • the treated and untreated cells are then compared by any suitable phenotypic criteria, including but not limited to microscopic analysis, viability testing, ability to replicate, histo logical examination, the level of a particular RNA or polypeptide associated with the cells, the level of enzymatic activity expressed by the cells or cell lysates, the interactions of the cells when exposed to infectious agents, such as HIN, and the ability of the cells to interact with other cells or compounds.
  • suitable phenotypic criteria including but not limited to microscopic analysis, viability testing, ability to replicate, histo logical examination, the level of a particular RNA or polypeptide associated with the cells, the level of enzymatic activity expressed by the cells or cell lysates, the interactions of the cells when exposed to infectious agents, such as HIN, and the ability of the cells to interact with other cells or compounds.
  • the transfected cells can be exposed to the agent to be tested and, before, during, or after treatment with the agent, the cells can be infected with a virus, such as HIN, and tested for any indication of susceptibility of the cells to viral infection, including, for example, susceptibility of the cells to cell-to-cell viral infection, replication of the virus, production of a viral protein, and/or syncytia formation following infection with the virus. Differences between treated and untreated cells indicate effects attributable to the candidate agent. Optimally, the agent has a greater effect on experimental cells than on control cells.
  • Appropriate host cells include, but are not limited to, eukaryotic cells, preferably mammalian cells. The choice of cell will at least partially depend on the nature of the assay contemplated.
  • a suitable host cell transfected with a polynucleotide of interest such that the polynucleotide is expressed (as used herein, expression includes transcription and/or translation) is contacted with an agent to be tested.
  • An agent would be tested for its ability to result in increased expression of mR ⁇ A and/or polypeptide.
  • Methods of making vectors and transfection are well known in the art. "Transfection” encompasses any method of introducing the endogenous sequence, including, for example, lipofection, transduction, 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.
  • reporter gene means a gene that encodes a gene product that can be identified (i.e., a reporter protein).
  • Reporter genes include, but are not limited to, alkaline phosphatase, chloramphenicol acetyltransferase, ⁇ -galactosidase, luciferase and green fluorescence protein (GFP). Identification methods for the products of reporter genes include, but are not limited to, enzymatic assays and fluorimetric assays.
  • Reporter genes and assays to detect their products are well known in the art and are described, for example in Ausubel et al. (1987) and periodic updates. Reporter genes, reporter gene assays, and reagent kits are also readily available from commercial sources. Examples of appropriate cells include, but are not limited to, fungal, yeast, mammalian, and other eukaryotic cells. A practitioner of ordinary skill will be well acquainted with techniques for transfecting eukaryotic cells, including the preparation of a suitable vector, such as a viral vector; conveying the vector into the cell, such as by electroporation; and selecting cells that have been transformed, such as by using a reporter or drug sensitivity element. The effect of an agent on transcription from the regulatory region in these constructs would be assessed through the activity of the reporter gene product.
  • expression could be decreased when it would normally be expressed.
  • An agent could accomplish this through a decrease in transcription rate and the reporter gene system described above would be a means to assay for this.
  • the host cells to assess such agents would be need to be permissive for expression.
  • Cells transcribing mRNA could be used to identify agents that specifically modulate the half-life of mRNA and/or the translation of mRNA. Such cells would also be used to assess the effect of an agent on the processing and/or post-translational modification of the polypeptide.
  • An agent could modulate the amount of polypeptide in a cell by modifying the turn-over (i.e., increase or decrease the half-life) of the polypeptide.
  • the specificity of the agent with regard to the mRNA and polypeptide would be determined by examining the products in the absence of the agent and by examining the products of unrelated mRNAs and polypeptides. Methods to examine mRNA half-life, protein processing, and protein turn-over are well know to those skilled in the art.
  • agents that modulate polypeptide function through the interaction with the polypeptide directly could block normal polypeptide-ligand interactions, if any, or could enhance or stabilize such interactions. Such agents could also alter a conformation of the polypeptide.
  • the effect of the agent could be determined using immunoprecipitation reactions. Appropriate antibodies would be used to precipitate the polypeptide and any protein tightly associated with it. By comparing the polypeptides immunoprecipitated from treated cells and from untreated cells, an agent could be identified that would augment or inhibit polypeptide-ligand interactions, if any.
  • Polypeptide-ligand interactions could also be assessed using cross-linking reagents that convert a close, but noncovalent interaction between polypeptides into a covalent interaction. Techniques to examine protein-protein interactions are well known to those skilled in the art. Techniques to assess protein conformation are also well known to those skilled in the art.
  • screening methods can involve in vitro methods, such as cell-free transcription or translation systems.
  • transcription or translation is allowed to occur, and an agent is tested for its ability to modulate function.
  • an in vitro transcription/translation system may be used for an assay that determines whether an agent modulates the translation of mRNA or a polynucleotide.
  • these systems are available commercially and provide an in vitro means to produce mRNA corresponding to a polynucleotide sequence of interest. After mRNA is made, it can be translated in vitro and the translation products compared. Comparison of translation products between an in vitro expression system that does not contain any agent (negative control) with an in vitro expression system that does contain an agent indicates whether the agent is affecting translation.
  • Comparison of translation products between control and test polynucleotides indicates whether the agent, if acting on this level, is selectively affecting translation (as opposed to affecting translation in a general, non-selective or non-specific fashion).
  • the modulation of polypeptide function can be accomplished in many ways including, but not limited to, the in vivo and in vitro assays listed above as well as in in vitro assays using protein preparations.
  • Polypeptides can be extracted and/or purified from natural or recombinant sources to create protein preparations.
  • An agent can be added to a sample of a protein preparation and the effect monitored; that is whether and how the agent acts on a polypeptide and affects its conformation, folding (or other physical characteristics), binding to other moieties (such as ligands), activity (or other functional characteristics), and/or other aspects of protein structure or functions is considered to have modulated polypeptide function.
  • a polypeptide is first recombinantly expressed in a prokaryotic or eukaryotic expression system as a native or as a fusion protein in which a polypeptide (encoded by a polynucleotide identified as described above) is conjugated with a well-characterized epitope or protein.
  • Recombinant polypeptide is then purified by, for instance, immunoprecipitation using appropriate antibodies or anti-epitope antibodies or by binding to immobilized ligand of the conjugate.
  • An affinity column made of polypeptide or fusion protein is then used to screen a mixture of compounds which have been appropriately labeled. Suitable labels include, but are not limited to fluorochromes, radioisotopes, enzymes and chemiluminescent compounds.
  • the unbound and bound compounds can be separated by washes using various conditions (e.g. high salt, detergent ) that are routinely employed by those skilled in the art.
  • Non-specific binding to the affinity column can be minimized by pre-clearing the compound mixture using an affinity column containing merely the conjugate or the epitope.
  • Similar methods can be used for screening for an agent(s) that competes for binding to polypeptides.
  • affinity chromatography there are other techniques such as measuring the change of melting temperature or the fluorescence anisotropy of a protein which will change upon binding another molecule.
  • a BIAcore assay using a sensor chip supplied by Pharmacia Biosensor, Stitt et al. (1995) Cell 80: 661-670) that is covalently coupled to polypeptide may be performed to determine the binding activity of different agents.
  • the in vitro screening methods of this invention include structural, or rational, drug design, in which the amino acid sequence, three- dimensional atomic structure or other property (or properties) of a polypeptide provides a basis for designing an agent which is expected to bind to a polypeptide.
  • the design and/or choice of agents in this context is governed by several parameters, such as side-by-side comparison of the structures of a human and homologous non-human primate polypeptides, the perceived function of the polypeptide target, its three-dimensional structure (if known or surmised), and other aspects of rational drug design.
  • Techniques of combinatorial chemistry can also be used to generate numerous permutations of candidate agents.
  • transgenic animal systems which are known in the art.
  • a secondary screen may comprise testing the agent(s) in an infectivity assay using mice and other animal models (such as rat), which are known in the art.
  • a cytotoxicity assay would be performed as a further corroboration that an agent which tested positive in a primary screen would be suitable for use in living organisms. Any assay for cytotoxicity would be suitable for this purpose, including, for example the MTT assay (Promega).
  • the invention also includes agents identified by the screening methods described herein. METHODS USEFUL FOR IDENTIFYING POSITIVELY SELECTED NON-HUMAN TRAITS
  • a non-human primate polynucleotide or polypeptide has undergone natural selection that resulted in a positive evolutionarily significant change (i.e., the non-human primate polynucleotide or polypeptide has a positive attribute not present in humans).
  • the positively selected polynucleotide or polypeptide may be associated with susceptibility or resistance to certain diseases or with other commercially relevant traits.
  • Examples of this embodiment include, but are not limited to, polynucleotides and polypeptides that have been positively selected in non-human primates, preferably chimpanzees, that may be associated with susceptibility or resistance to infectious diseases, cancer, or acne or may be associated with aesthetic conditions of interest to humans, such as hair growth or muscle mass.
  • An example of this embodiment includes polynucleotides and polypeptides associated with the susceptibility or resistance to HIV progression to AIDS. The present invention can thus be useful in gaining insight into the molecular mechanisms that underlie resistance to HIV infection progressing to development of AIDS, providing information that can also be useful in discovering and/or designing agents such as drugs that prevent and/or delay development of AIDS.
  • Commercially relevant examples include, but are not limited to, polynucleotides and polypeptides that are positively selected in non-human primates that may be associated with aesthetic traits, such as hair growth, acne, or muscle mass.
  • the invention provides methods for identifying a polynucleotide sequence encoding a polypeptide, wherein said polypeptide may be associated with a medically relevant positive evolutionarily significant change.
  • the positive evolutionarily significant change can be found in humans or in non-human primates, but the positively selected non-human primate evolutionarily significant change will be described first herein.
  • the method comprises the steps of: (a) comparing human protein-coding nucleotide sequences to protein-coding nucleotide sequences of a non-human primate; and (b) selecting a human polynucleotide sequence that contains at least one nucleotide change as compared to corresponding sequence of the non-human primate, wherein said change is evolutionarily significant.
  • the sequences identified by this method may be further characterized and/or analyzed for their possible association with biologically or medically relevant functions unique or enhanced in humans.
  • Also provided in the present invention is a method for identifying a positive evolutionarily significant change within human protein-coding nucleotide sequences, comprising the steps of: (a) comparing human protein-coding nucleotide sequences to corresponding sequences of a non-human primate; and (b) selecting a human polynucleotide sequence that contains at least one nucleotide change as compared to the corresponding sequence of the non-human primate, wherein said change is evolutionarily significant.
  • This invention specifically provides methods for identifying human polynucleotide and polypeptide sequences that may be associated with unique or enhanced functional capabilities of the human, for example, brain function or longer life span. More particularly, these methods identify those genetic sequences that may be associated with capabilities that are unique or enhanced in humans, including, but not limited to, brain functions such as high capacity information processing, storage and retrieval capabilities, creativity, and language abilities. Moreover, these methods identify those sequences that may be associated to other brain functional features with respect to which the human brain performs at enhanced levels as compared to other non-human primates; these differences may include brain-mediated emotional response, locomotion, pain/pleasure sensation, olfaction, temperament and longer life span In this method, the general methods of the invention are applied as described above.
  • the methods described herein entail (a) comparing human protein-coding polynucleotide sequences to that of a non-human primate; and (b) selecting those human protein-coding polynucleotide sequences having evolutionarily significant changes that may be associated with unique or enhanced functional capabilities of the human as compared to that of the non-human primate.
  • the human sequence includes the evolutionarily significant change (i.e., the human sequence differs from more than one non-human primate species sequence in a manner that suggests that such a change is in response to a selective pressure).
  • the identity and function of the protein encoded by the gene that contains the evolutionarily significant change is characterized and a determination is made whether or not the protein can be involved in a unique or enhanced human function. If the protein is involved in a unique or enhanced human function, the information is used in a manner to identify agents that can supplement or otherwise modulate the unique or enhanced human function.
  • identifying the genetic (i.e., nucleotide sequence) differences underlying the functional uniqueness of human brain may provide a basis for designing agents that can modulate human brain functions and/or help correct functional defects. These sequences could also be used in developing diagnostic reagents and/or biomedical research tools.
  • the invention also provides methods for a large-scale comparison of human brain protein-coding sequences with that from a non-human primate.
  • the identified human sequence changes can be used in establishing a database of candidate human genes that may be involved in human brain function. Candidates are ranked as to the likelihood that the gene is responsible for the unique or enhanced functional capabilities found in the human brain compared to chimpanzee or other non-human primates. Moreover, the database not only provides an ordered collection of candidate genes, it also provides the precise molecular sequence differences that exist between human and chimpanzee (and other non-human primates), and thus defines the changes that underlie the functional differences. This information can be useful in the identification of potential sites on the protein that may serve as useful targets for pharmaceutical agents.
  • the present invention also provides methods for correlating an evolutionarily significant nucleotide change to a brain functional capability that is unique or enhanced in humans, comprising (a) identifying a human nucleotide sequence according to the methods described above; and (b) analyzing the functional effect of the presence or absence of the identified sequence in a model system.
  • the putative function can be assayed in appropriate in vitro assays using transiently or stably transfected mammalian cells in culture, or using mammalian cells transfected with an antisense clone to inhibit expression of the identified polynucleotide to assess the effect of the absence of expression of its encoded polypeptide.
  • Studies such as one-hybrid and two-hybrid studies can be conducted to determine, for example, what other macromolecules the polypeptide interacts with.
  • Drosophila can be used for various functional assays, including behavioral studies. The appropriate studies depend on the nature of the identified polynucleotide and the polypeptide encoded within the polynucleotide, and would be obvious to those skilled in the art. Description of the AIDS Embodiment (an example of a positively selected non-human trait)
  • the AIDS (Acquired Immune Deficiency Syndrome) epidemic has been estimated to threaten 30 million people world-wide (UNAIDS/WHO, 1998, "Report on the global HIV/ AIDS epidemic”).
  • UnAIDS/WHO 1998, "Report on the global HIV/ AIDS epidemic”
  • Well over a million people are infected in developed countries, and in parts of sub-Saharan Africa, 1 in 4 adults now carries the virus (UNAIDS/WHO, 1998).
  • Baiter and Cohen 1998 Science 281 :159-160; Baltimore and Heilman (1998) Scientific Am. 279:98-103.
  • Further complicating the development of therapeutics is the rapid mutation rate of HIV (the human immunodeficiency virus which is responsible for AIDS), which generates rapid changes in viral proteins.
  • protein coding polynucleotides may contain sequence changes that are found in chimpanzees (as well as other AIDS-resistant primates) but not in humans, likely as a result of positive adaptive selection during evolution. Furthermore, such evolutionarily significant changes in polynucleotide and polypeptide sequences may be attributed to an AIDS-resistant non-human primate's (such as chimpanzee) ability to resist development of AIDS.
  • the methods of this invention employ selective comparative analysis to identify candidate genes which may be associated with susceptibility or resistance to AIDS, which may provide new host targets for therapeutic intervention as well as specific information on the changes that evolved to confer resistance.
  • the invention also provides screening methods using the sequences and structural differences identified.
  • This invention provides methods for identifying human polynucleotide and polypeptide sequences that may be associated with susceptibility to post-infection development of AIDS.
  • the invention also provides methods for identifying polynucleotide and polypeptide sequences from an AIDS-resistant non- human primate (such as chimpanzee) that may be associated with resistance to development of AIDS.
  • Identifying the genetic (i.e., nucleotide sequence) and the resulting protein structural and biochemical differences underlying susceptibility or resistance to development of AIDS will likely provide a basis for discovering and/or designing agents that can provide prevention and/or therapy for HIV infection progressing to AIDS. These differences could also be used in developing diagnostic reagents and/or biomedical research tools. For example, identification of proteins which confer resistance may allow development of diagnostic reagents or biomedical research tools based upon the disruption of the disease pathway of which the resistant protein plays a part.
  • the methods described herein entail (a) comparing human protein- coding polynucleotide sequences to that of an AIDS resistant non-human primate (such as chimpanzee), wherein the human protein coding polynucleotide sequence is associated with development of AIDS; and (b) selecting those human protein-coding polynucleotide sequences having evolutionarily significant changes that may be associated with susceptibility to development of AIDS.
  • an AIDS resistant non-human primate such as chimpanzee
  • the methods entail (a) comparing human protein-coding polynucleotide sequences to that of an AIDS-resistant non-human primate (such as chimpanzee), wherein the human protein coding polynucleotide sequence is associated with development of AIDS; and (b) selecting those non-human primate protein-coding polynucleotide sequences having evolutionarily significant changes that may be associated with resistance to development of AIDS.
  • an AIDS-resistant non-human primate such as chimpanzee
  • the methods described herein can be applied to other infectious diseases.
  • the methods could be used in a situation in which a non- human primate is known or believed to have harbored the infectious disease for a significant period (i.e., a sufficient time to have allowed positive selection) and is resistant to development of the disease.
  • the invention provides methods for identifying a polynucleotide sequence encoding a polypeptide, wherein said polypeptide may be associated with resistance to development of an infectious disease, comprising the steps of: (a) comparing infectious disease-resistant non-human primate protein coding sequences to human protein coding sequences, wherein the human protein coding sequence is associated with development of the infectious disease; and (b) selecting an infectious disease-resistant non-human primate sequence that contains at least one nucleotide change as compared to the corresponding human sequence, wherein the nucleotide change is evolutionarily significant.
  • the invention provides methods for identifying a human polynucleotide sequence encoding a polypeptide, wherein said polypeptide may be associated with susceptibility to development of an infectious disease, comprising the steps of: (a) comparing human protein coding sequences to protein- coding polynucleotide sequences of an infectious disease-resistant non-human primate, wherein the human protein coding sequence is associated with development of the infectious disease; and (b) selecting a human polynucleotide sequence that contains at least one nucleotide change as compared to the corresponding sequence of an infectious disease-resistant non-human primate, wherein the nucleotide change is evolutionarily significant.
  • human sequences to be compared with a homologue from an AIDS-resistant non-human primate are selected based their known or implicated association with HIV propagation (i.e., replication), dissemination and/or subsequent progression to AIDS.
  • Such knowledge is obtained, for example, from published literature and/or public databases (including sequence databases such as
  • GenBank Because the pathway involved in development of AIDS (including viral replication) involves many genes, a number of suitable candidates may be tested using the methods of this invention.
  • Table 1 contains a exemplary list of genes to be examined. The sequences are generally known in the art. Table 1: Sample List of Human Genes to be/have been Examined Gene Function
  • PCD promoter bcl-2 apoptosis inhibitor lck tyrosine kinase MAPK (mitogen activated protein kinase) protein kinase
  • Immune system Mac-1 ⁇ (equivalent to LFA-1 ⁇ ) Immune system CXCR4 chemokine receptor CCR5 chemokine receptor MlP-l ⁇ chemokine MlP-l ⁇ chemokine RANTES chemokine
  • Aligned protein-coding sequences of human and an AIDS resistant non- human primate such as chimpanzee are analyzed to identify nucleotide sequence differences at particular sites.
  • the detected sequence changes are generally, and preferably, initially checked for accuracy as described above.
  • the evolutionarily significant nucleotide changes, which are detected by molecular evolution analysis such as the K A /K S analysis, can be further assessed to determine whether the non- human primate gene or the human gene has been subjected to positive selection. For example, the identified changes can be tested for presence/absence in other AIDS- resistant non-human primate sequences.
  • sequences with at least one evolutionarily significant change between human and one AIDS-resistant non-human primate can be used as primers for PCR analysis of other non-human primate protein- coding sequences, and resulting polynucleotides are sequenced to see whether the same change is present in other non-human primates.
  • These comparisons allow further discrimination as to whether the adaptive evolutionary changes are unique to the AIDS-resistant non-human primate (such as chimpanzee) as compared to other non- human primates. For example, a nucleotide change that is detected in chimpanzee but not other primates more likely represents positive selection on the chimpanzee gene.
  • Other non-human primates used for comparison can be selected based on their phylogenetic relationships with human.
  • Closely related primates can be those within the hominoid sublineage, such as chimpanzee, bonobo, gorilla, and orangutan.
  • Non- human primates can also be those that are outside the hominoid group and thus not so closely related to human, such as the Old World monkeys and New World monkeys.
  • Statistical significance of such comparisons may be determined using established available programs, e.g., t-test as used by Messier and Stewart (1997) Nature 385:151-154.
  • sequences with significant changes can be used as probes in genomes from different humans to see whether the sequence changes are shared by more than one individual. For example, certain individuals are slower to progress to
  • AIDS slow progressers
  • comparison (a) between a chimpanzee sequence and the homologous sequence from the slow-progresser human individual and/or (b) between an AIDS-susceptible individual and a slow-progresser individual would be of interest.
  • Gene sequences from different human populations can be obtained from databases made available by, for example, the human genome diversity project or, alternatively, from direct sequencing of PCR-amplified DNA from a number of unrelated, diverse human populations.
  • the presence of the identified changes in human slow progressers would further indicate the evolutionary significance of the changes.
  • the following examples are provided to further assist those of ordinary skill in the art. Such examples are intended to be illustrative and therefore should not be regarded as limiting the invention.
  • a number of exemplary modifications and variations are described in this application and others will become apparent to those of skill in this art. Such variations are considered to fall within the scope of the invention as described and claimed herein.
  • a chimpanzee cDNA library is constructed using chimpanzee tissue.
  • RNA Total RNA is extracted from the tissue (RNeasy kit, Quiagen; RNAse-free Rapid Total RNA kit, 5 Prime-3 Prime, Inc.) and the integrity and purity of the RNA are determined according to conventional molecular cloning methods.
  • Poly A+ RNA is isolated (Mini-Oligo(dT) Cellulose Spin Columns, 5 Prime—3 Prime, Inc.) and used as template for the reverse-transcription of cDNA with oligo (dT) as a primer.
  • the synthesized cDNA is treated and modified for cloning using commercially available kits. Recombinants are then packaged and propagated in a host cell line. Portions of the packaging mixes are amplified and the remainder retained prior to amplification.
  • the library can be normalized and the numbers of independent recombinants in the library is determined.
  • Suitable primers based on a candidate human gene are prepared and used for
  • PCR amplification of chimpanzee cDNA either from a cDNA library or from cDNA prepared from mRNA Selected chimpanzee cDNA clones from the cDNA library are sequenced using an automated sequencer, such as an ABI 377. Commonly used primers on the cloning vector such as the Ml 3 Universal and Reverse primers are used to carry out the sequencing. For inserts that are not completely sequenced by end sequencing, dye-labeled terminators are used to fill in remaining gaps.
  • the detected sequence differences are initially checked for accuracy, for example by finding the points where there are differences between the chimpanzee and human sequences; checking the sequence fluorogram (chromatogram) to determine if the bases that appear unique to human correspond to strong, clear signals specific for the called base; checking the human hits to see if there is more than one human sequence that corresponds to a sequence change; and other methods known in the art, as needed.
  • Multiple human sequence entries for the same gene that have the same nucleotide at a position where there is a different chimpanzee nucleotide provides independent support that the human sequence is accurate, and that the chimpanzee/human difference is real. Such changes are examined using public database information and the genetic code to determine whether these DNA sequence changes result in a change in the amino acid sequence of the encoded protein.
  • the sequences can also be examined by direct sequencing of the encoded protein.
  • K A /K S The chimpanzee and human sequences under comparison are subjected to K A /K S analysis.
  • publicly available computer programs such as Li 93 and INA, are used to determine the number of non-synonymous changes per site (K A ) divided by the number of synonymous changes per site (K s ) for each sequence under study as described above.
  • Full-length coding regions or partial segments of a coding region can be used.
  • K A /K S ratio the more likely that a sequence has undergone adaptive evolution.
  • Statistical significance of K A /K S values is determined using established statistic methods and available programs such as the t-test.
  • sequence under study can be compared in multiple chimpanzee individuals and in other non- human primates, e.g., gorilla, orangutan, bonobo. These comparisons allow further discrimination as to whether the adaptive evolutionary changes are unique to the human lineage compared to other non-human primates.
  • sequences can also be examined by direct sequencing of the gene of interest from representatives of several diverse human populations to assess to what degree the sequence is conserved in the human species.
  • intercellular adhesion molecules ICAM-1, ICAM-2 and ICAM-3 have been shown to have been strongly positively selected.
  • the ICAM molecules are involved in several immune response interactions and are known to play a role in progression to AIDS in HIV infected humans.
  • the ICAM proteins members of the Ig superfamily, are ligands for the integrin leukocyte associated function 1 molecule (LFA-1). Makgoba et al. (1988)
  • LFA-1 is expressed on the surface of most leukocytes
  • ICAMs are expressed on the surface of both leukocytes and other cell types. Larson et al. (1989) J Cell Biol. 108:703-712. ICAM and LFA-1 proteins are involved in several immune response interactions, including T-cell function, and targeting of leukocytes to areas of inflammation. Larson et al. (1989).
  • Total RNA was prepared using either the RNeasy kit (Qiagen), or the RNAse- free Rapid Total RNA kit (5 Prime - 3 Prime, Inc.) from primate tissues (chimpanzee brain and blood, gorilla blood and spleen, orangutan blood) or from cells harvested from the following B lymphocyte cell lines: CARL (chimpanzee), ROK (gorilla), and PUTI (orangutan).
  • mRNA was isolated from total RNA using the Mini-Oligo(dT) Cellulose Spin Columns (5 Prime - 3 Prime, Inc.).
  • cDNA was synthesized from mRNA with oligo dT and/or random priming using the cDNA Synthesis Kit (Stratagene).
  • the protein-coding region of the primate ICAM-1 gene was amplified from cDNA using primers (concentration ⁇ lOO nmole/ ⁇ l) designed by hand from the published human sequence.
  • PCR conditions for ICAM-1 amplification were 94°C initial pre-melt (4 min), followed by 35 cycles of 94°C (15 sec), 58°C (1 min 15 sec), 72°C (1 min 15 sec), and a final 72°C extension for 10 minutes.
  • PCR was accomplished using Ready-to-Go PCR beads (Amersham Pharmacia Biotech) in a 50 microliter total reaction volume. Appropriately-sized products were purified from agarose gels using the QiaQuick Gel Extraction kit (Qiagen).
  • Syncytia may simply be scavenged from the body too quickly to be detected. Fouchier et ⁇ /. (1996) Virology 219:87-95. Syncytia-mediated loss of CD4 + cells in vivo has been speculated to occur; this could contribute directly to compromise of the immune system, leading to opportunistic infection and full-blown AIDS. Sodrosky et al. (1986) Nature 322:470-474; Hildreth and Orentas (1989)
  • a sequence identified by the methods of this invention may be further tested and characterized by cell transfection experiments.
  • human cells in culture when transfected with a chimpanzee polynucleotide identified by the methods described herein (such as ICAM-1 (or ICAM-2 or ICAM-3); see below), could be tested for reduced viral dissemination and/or propagation using standard assays in the art, and compared to control cells.
  • Other indicia may also be measured, depending on the perceived or apparent functional nature of the polynucleotide/polypeptide to be tested.
  • ICAM-1 or ICAM-2 or ICAM-3
  • syncytia formation may be measured and compared to control (untransfected) cells. This would test whether the resistance arises from prevention of syncytia formation in infected cells.
  • Cells which are useful in characterizing sequences identified by the methods of this invention and their effects on cell-to-cell infection by HIV-1 are human T-cell lines which are permissive for infection with HIV-1 , including, e.g., H9 and HUT78 cell lines, which are available from the ATCC.
  • ICAM-1 (or ICAM-2 or ICAM-3) cDNA (or any cDNA identified by the methods described herein) can be cloned into an appropriate expression vector.
  • the cloned ICAM-1 (or ICAM-2 or ICAM-3) coding region is operably linked to a promoter which is active in human T cells, such as, for example, an IL-2 promoter.
  • a promoter which is active in human T cells
  • an ICAM-1 (or ICAM-2 or ICAM-3) cDNA can be placed under transcriptional control of a strong constitutive promoter, or an inducible promoter. Expression systems are well known in the art, as are methods for introducing an expression vector into cells.
  • an expression vector comprising an ICAM-1 (or ICAM-2 or ICAM-3) cDNA can be introduced into cells by DEAE-dextran or by electroporation, or any other known method.
  • the cloned ICAM-1 (or ICAM-2 or ICAM-3) molecule is then expressed on the surface of the cell.
  • Determination of whether an ICAM-1 (or ICAM-2 or ICAM- 3) cDNA is expressed on the cell surface can be accomplished using antibody (ies) specific for ICAM-1 (or ICAM-2 or ICAM-3). In the case of chimpanzee ICAM-1 (or
  • ICAM-2 or ICAM-3) expressed on the surface of human T cells an antibody which distinguishes between chimpanzee and human ICAM-1 (or ICAM-2 or ICAM-3) can be used.
  • This antibody can be labeled with a detectable label, such as a fluorescent dye.
  • Cells expressing chimpanzee ICAM-1 (or ICAM-2 or ICAM-3) on their surfaces can be detected using fluorescence-activated cell sorting and the anti-ICAM-1 (or
  • ICAM-2 or ICAM-3) antibody appropriately labeled, using well-established techniques.
  • Transfected human cells expressing chimpanzee ICAM-1 (or ICAM-2 or ICAM-3) on their cell surface can then be tested for syncytia formation, and/or for HIV replication, and/or for number of cells infected as an index of cell-to-cell infectivity.
  • the chimpanzee ICAM-1 (or ICAM-2 or ICAM-3)-expressing cells can be infected with HIV-1 at an appropriate dose, for example tissue culture infectious dose 50, i.e., a dose which can infect 50%o of the cells.
  • Cells can be plated at a density of about 5 x 10 5 cells/ml in appropriate tissue culture medium, and, after infection, monitored for syncytia formation, and/or viral replication, and/or number of infected cells in comparison to control, uninfected cells.
  • Cells which have not been transfected with chimpanzee ICAM-1 (or ICAM-2 or ICAM-3) also serve as controls.
  • Syncytia formation is generally observed in HIV-1 -infected cells (which are not expressing chimpanzee ICAM-1 (or ICAM-2 or ICAM-3)) approximately 10 days post-infection.
  • cell supernatants can be assayed for the presence and amount of p24 antigen.
  • Any assay method to detect p24 can be used, including, for example, an ELISA assay in which rabbit anti-p24 antibodies are used as capture antibody, biotinylated rabbit anti-p24 antibodies serve as detection antibody, and the assay is developed with avidin-horse radish peroxidase.
  • any known method including indirect immunofluorescence methods, can be used.
  • indirect immunofluorescence methods human HIV-positive serum can be used as a source of anti-HIV antibodies to bind to infected cells. The bound antibodies can be detected using FITC-conjugated anti-human IgG, the cells visualized by fluorescence microscopy and counted.
  • Another method for assessing the role of a molecule such as ICAM-1 involves successive infection of cells with HIV.
  • Human cell lines preferably those that do not express endogenous ICAM (although cell lines that do express endogenous ICAM may also be used), are transfected with either human or chimpanzee ICAM -1 or -2 or -3.
  • HIV is collected from the supernatant of HIV-infected human ICAM-1 (or ICAM-2 or ICAM-3)-expressing cells and used to infect chimpanzee ICAM-1 (or ICAM-2 or ICAM-3 )-expressing cells or human ICAM-1 (or ICAM-2 or ICAM-3)-expressing cells.
  • the initial infectivity would be expected to be much lower than in the first set of experiments, if ICAM-1 (or ICAM-2 or ICAM-3) is involved in susceptibility to HIV progression. After several rounds of replication, the cell to cell infectivity would be expected to increase.
  • the identified human sequences can be used in establishing a database of candidate human genes that may be involved in conferring, or contributing to, AIDS susceptibility or resistance. Moreover, the database not only provides an ordered collection of candidate genes, it also provides the precise molecular sequence differences that exist between human and an AIDS-resistant non-human primate (such as chimpanzee) and thus defines the changes that underlie the functional differences.
  • binding constants are being determined. Should the binding constants prove lower in chimpanzee ICAM-1, it is possible to devise small molecule agents to mimic (by way of steric hindrance) the change in binding constants as a potential therapeutic strategy for HIV-infected humans. Similarly, stronger binding constants, if observed for chimpanzee ICAM-1 , will suggest alternative strategies for developing therapeutic interventions for HIV-1 infected humans.
  • ICAM-3 In the case of ICAM-3, a critical amino acid residue replacement from proline (observed in seven humans) to glutamine (observed in three chimpanzees) is predicted from our modeling studies to significantly change the positional angle between domains 2 and 3 of human and chimpanzee ICAM-3.
  • the human protein displays an acute angle at this juncture.
  • Loss of this sharp angle (bend) is predicted to render chimpanzee ICAM-3 less easily packaged into HIV-1 virions (In infected humans, after ICAMs are packaged into HIV virions, cell-to-cell infectivity dramatically increases.
  • a small molecule therapeutic intervention whereby binding of a suitably- designed small molecule to the human proline residue causes (as a result of steric hindrance) the human ICAM-1 protein to mimic the larger (i.e., less-acute) angle of chimpanzee ICAM-3 is possible.
  • Conservation between the 2 proteins of the critical binding residues (and the general resemblance of immune responses between humans and chimpanzees) argues that alteration of this angle will not compromise the basic function of human ICAM-3.
  • the human ICAM-3 protein would be rendered resistant to packaging into HIV virions, thus mimicking (in HIV-1 infected humans) the postulated pathway by which infected chimpanzees resist progression to AIDS.
  • MlP-l ⁇ is a chemokine that has been shown to suppress HIV-1 replication in human cells in vitro (Cocchi, F. et al, 1995 Science 270:1811-1815).
  • the chimpanzee homologue of the human MIP-1 ⁇ gene was PCR-amplified and sequenced. Calculation of the K A /K S ratio (2.1, P ⁇ 0.05) and comparison to the gorilla homologue reveals that the chimpanzee gene has been positively-selected.
  • the nature of the chimpanzee amino acid replacements is being examined to determine how to exploit the chimpanzee protein for therapeutic intervention.
  • the human gene 17- ⁇ hydroxysteroid dehydrogenase type IV, codes for a protein known to degrade the two most potent estrogens, ⁇ -estradiol, and 5-diol (Adamski, J. et al. 1995 Biochem J. 311:437-443).
  • Estrogen-related cancers including, for example, breast and prostate cancers) account for some 40%> of human cancers.
  • chimpanzees are resistant to tumorigenesis, especially those that are estrogen-related.
  • This protein may have been positively-selected in chimpanzees to allow more efficient degradation of estrogens, thus conferring upon chimpanzees resistance to such cancers. If so, the specific amino acid replacements observed in the chimpanzee protein may supply important information for therapeutic intervention in human cancers.
  • a chimpanzee brain cDNA library is constructed using chimpanzee brain tissue.
  • the chimpanzee brain tissue can be obtained after natural death so that no killing of animal is necessary for this study. In order to increase the chance of obtaining intact mRNAs expressed in brain, however, the brain is obtained as soon as possible after the animal's death. Preferably, the weight and age of the animal are determined prior to death.
  • the brain tissue used for constructing a cDNA library is preferably the whole brain in order to maximize the inclusion of mRNA expressed in the entire brain. Brain tissue is dissected from the animal following standard surgical procedures.
  • Total RNA is extracted from the brain tissue and the integrity and purity of the RNA are determined according to conventional molecular cloning methods.
  • Poly A+ RNA is selected and used as template for the reverse-transcription of cDNA with oligo (dT) as a primer.
  • the synthesized cDNA is treated and modified for cloning using commercially available kits. Recombinants are then packaged and propagated in a host cell line. Portions of the packaging mixes are amplified and the remainder retained prior to amplification.
  • the library can be normalized and the numbers of independent recombinants in the library is determined.
  • Randomly selected chimpanzee brain cDNA clones from the cDNA library are sequenced using an automated sequencer, such as the ABI 377. Commonly used primers on the cloning vector such as the Ml 3 Universal and Reverse primers are used to carry out the sequencing. For inserts that are not completely sequenced by end sequencing, dye-labeled terminators are used to fill in remaining gaps.
  • the resulting chimpanzee sequences are compared to human sequences via database searches, e.g., BLAST searches.
  • the high scoring "hits," i.e., sequences that show a significant (e.g., >80%o) similarity after BLAST analysis, are retrieved and analyzed.
  • the two homologous sequences are then aligned using the alignment program CLUSTAL V developed by Higgins et al. Any sequence divergence, including nucleotide substitution, insertion and deletion, can be detected and recorded by the alignment.
  • the detected sequence differences are initially checked for accuracy by finding the points where there are differences between the chimpanzee and human sequences; checking the sequence fluorogram (chromatogram) to determine if the bases that appear unique to human correspond to strong, clear signals specific for the called base; checking the human hits to see if there is more than one human sequence that corresponds to a sequence change; and other methods known in the art as needed.
  • Multiple human sequence entries for the same gene that have the same nucleotide at a position where there is a different chimpanzee nucleotide provides independent support that the human sequence is accurate, and that the chimpanzee/human difference is real.
  • Such changes are examined using public database information and the genetic code to determine whether these DNA sequence changes result in a change in the amino acid sequence of the encoded protein.
  • the sequences can also be examined by direct sequencing of the encoded protein.
  • K A /K S The chimpanzee and human sequences under comparison are subjected to K A /K S analysis.
  • publicly available computer programs such as Li 93 and INA, are used to determine the number of non-synonymous changes per site (K A ) divided by the number of synonymous changes per site (K s ) for each sequence under study as described above.
  • K A /Ks This ratio, K A /Ks, has been shown to be a reflection of the degree to which adaptive evolution, i.e., positive selection, has been at work in the sequence under study.
  • full-length coding regions have been used in these comparative analyses. However, partial segments of a coding region can also be used effectively.
  • the higher the K A /K S ratio the more likely that a sequence has undergone adaptive evolution.
  • Statistical significance of K A /K S values is determined using established statistic methods and available programs such as the t-test. Those genes showing statistically high K A /K S ratios between chimpanzee
  • sequence under study can be compared in other non-human primates, e.g., gorilla, orangutan, bonobo. These comparisons allow further discrimination as to whether the adaptive evolutionary changes are unique to the human lineage compared to other non-human primates.
  • the sequences can also be examined by direct sequencing of the gene of interest from representatives of several diverse human populations to assess to what degree the sequence is conserved in the human species.
  • Human brain nucleotide sequences containing evolutionarily significant changes are further characterized in terms of their molecular and genetic properties, as well as their biological functions.
  • the identified coding sequences are used as probe to perform in situ mRNA hybridization that reveals the expression pattern of the gene, either or both in terms of what tissues and cell types in which the sequences are expressed, and when they are expressed during the course of development or during cell cycle. Sequences that are expressed in brain may be better candidates as being associated with important human brain functions.
  • the putative gene with the identified sequences are subjected to a homologue searching in order to determine what functional classes the sequences belong to.
  • the identified human sequence changes may be useful in estimating the functional consequence of the change.
  • a database of candidate genes can be generated. Candidates are ranked as to the likelihood that the gene is responsible for the unique or enhanced abilities found in the human brain compared to chimpanzee or other non-human primates, such as high capacity information processing, storage and retrieval capabilities, language abilities, as well as others. In this way, this approach provides a new strategy by which such genes can be identified.
  • the database not only provides an ordered collection of candidate genes, it also provides the precise molecular sequence differences that exist between human and chimpanzee (and other non-human primates), and thus defines the changes that underlie the functional differences.
  • LTP long term potentiation
  • EXAMPLE 14 Identification of Positive Selection in a Human Tyrosine Kinase Gene
  • a human gene (GenBank Acc.# ABO 14541), expressed in brain has been identified, that has been positively-selected as compared to its chimpanzee homologue.
  • This gene which codes for a tyrosine kinase, is homologous to a well-characterized mouse gene (GenBank Acc.# AF011908) whose gene product, called AATYK, is known to trigger apoptosis (Gaozza, E. et al. 1997 Oncogene 15:3127-3135).
  • AATYK gene product
  • the tyrosine kinase domain of this protein is highly conserved between mouse, chimpanzee, and human (as are most tyrosine kinases). Interestingly, however, the region of the protein to which signaling proteins bind has been positively-selected in humans, but strongly conserved in both chimpanzees and mice. The region of the human protein to which signaling proteins bind has not only been positively-selected as a result of point nucleotide mutation, but additionally displays duplication of several SH2 binding domains that exist only as single copies in mouse and chimpanzee.

Abstract

L'invention concerne des techniques permettant d'identifier des séquences de polynucléotides et de polypeptides chez des primates humains/non humains, qui peuvent être associées à un état physiologique tel qu'une maladie (y compris la susceptibilité (chez un humain) ou la résistance (chez un chimpanzé) au développement du syndrome d'immunodéficience acquis (AIDS). Ces techniques utilisent la comparaison de séquences de primate humain et non humain au moyen de méthodes statistiques. Les séquences ainsi identifiées peuvent être utiles comme cibles thérapeutiques hôtes et/ou comme tests de criblage.
PCT/US1999/020209 1998-09-02 1999-09-01 Techniques permettant d'identifier des sequences de polynucleotides et de polypeptides associees a des etats physiologiques et medicaux WO2000012764A1 (fr)

Priority Applications (10)

Application Number Priority Date Filing Date Title
AU58058/99A AU5805899A (en) 1998-09-02 1999-09-01 Methods to identify polynucleotide and polypeptide sequences which may be associated with physiological and medical conditions
DE60036645T DE60036645T2 (de) 1999-08-05 2000-08-01 Methoden zur identifizierung evolutionär signifikanter änderungen in polynukleotid- und polypeptidsequenzen in domestizierten pflanzen und tieren
CA002390094A CA2390094A1 (fr) 1999-08-05 2000-08-01 Procedes pour identifier des changements evolutionnaires importants dans des sequences de polynucleotides et de polypeptides chez des plantes et des animaux domestiques
ES00960208T ES2292467T3 (es) 1999-08-05 2000-08-01 Procedimiento para identificar cambios significativos desde el punto de vista evolutivo en secuencias de polinucleotidos y de polipeptidos en plantas y animales domesticados.
AT00960208T ATE374834T1 (de) 1999-08-05 2000-08-01 Methoden zur identifizierung evolutionär signifikanter änderungen in polynukleotid- und polypeptidsequenzen in domestizierten pflanzen und tieren
JP2001515335A JP2004500036A (ja) 1999-08-05 2000-08-01 栽培（家畜化）された植物および動物中のポリヌクレオチドおよびポリペプチド配列における進化的に有意な変化を同定する方法
AU71399/00A AU784869B2 (en) 1999-08-05 2000-08-01 Methods to identify evolutionarily significant changes in polynucleotide and polypeptide sequences in domesticated plants and animals
DK00960208T DK1250449T3 (da) 1999-08-05 2000-08-01 Fremgangsmåde til identifikation af evolutionært signifikante ændringer i polynucleotid- og polypeptidsekvenser hos domesticerede planter og dyr
PCT/US2000/040517 WO2001011088A2 (fr) 1999-08-05 2000-08-01 Procedes pour identifier des changements evolutionnaires importants dans des sequences de polynucleotides et de polypeptides chez des plantes et des animaux domestiques
EP00960208A EP1250449B1 (fr) 1999-08-05 2000-08-01 Procedes pour identifier des changements evolutionnaires importants dans des sequences de polynucleotides et de polypeptides chez des plantes et des animaux domestiques

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US9898798P 1998-09-02 1998-09-02
US60/098,987 1998-09-02
USPCT/US99/01964 1999-01-29
PCT/US1999/001964 WO1999039006A2 (fr) 1998-01-30 1999-01-29 Techniques d'identification de sequences de polynucleotides et de polypeptides pouvant etre associees a des etats physiologiques et medicaux

Publications (2)

Publication Number Publication Date
WO2000012764A1 WO2000012764A1 (fr) 2000-03-09
WO2000012764A9 true WO2000012764A9 (fr) 2000-06-22

Family

ID=22271867

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1999/020209 WO2000012764A1 (fr) 1998-09-02 1999-09-01 Techniques permettant d'identifier des sequences de polynucleotides et de polypeptides associees a des etats physiologiques et medicaux

Country Status (1)

Country Link
WO (1) WO2000012764A1 (fr)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6866996B1 (en) 1998-01-30 2005-03-15 Evolutionary Genomics, Llc Methods to identify polynucleotide and polypeptide sequences which may be associated with physiological and medical conditions
US7247425B2 (en) 1998-01-30 2007-07-24 Evolutionary Genomics, Llc Methods to identify polynucleotide and polypeptide sequences which may be associated with physiological and medical conditions
EP1649067A4 (fr) * 2003-06-30 2007-01-03 Evolutionary Genomics Llc Methodes d'identification de sequences polynucleotidiques et polypeptidiques pouvant etre associees a des troubles physiologiques ou medicaux

Also Published As

Publication number Publication date
WO2000012764A1 (fr) 2000-03-09

Similar Documents

Publication Publication Date Title
US7462460B2 (en) Methods for identifying agents that increase the p44 function of microtubule assembly or resistance to HCV infection
AU769931B2 (en) Methods to identify polynucleotide and polypeptide sequences which may be associated with physiological and medical conditions
Lahr et al. Reducing the impact of PCR-mediated recombination in molecular evolution and environmental studies using a new-generation high-fidelity DNA polymerase
US20090304653A1 (en) Methods to identify polynucleotide and polypeptide sequences which may be associated with physiological and medical conditions
AU2003245488A8 (en) Functional sites
US6743580B2 (en) Methods for producing transgenic plants containing evolutionarily significant polynucleotides
AU2001275303B2 (en) Methods to identify polynucleotide and polypeptide sequences which may be associated with physiological and medical conditions
US20080003607A1 (en) Methods to identify polynucleotide and polypeptide sequences which may be associated with physiological and medical conditions
AU2001275303A1 (en) Methods to identify polynucleotide and polypeptide sequences which may be associated with physiological and medical conditions
WO1999039006A2 (fr) Techniques d'identification de sequences de polynucleotides et de polypeptides pouvant etre associees a des etats physiologiques et medicaux
US7247425B2 (en) Methods to identify polynucleotide and polypeptide sequences which may be associated with physiological and medical conditions
WO2000012764A9 (fr) Techniques permettant d'identifier des sequences de polynucleotides et de polypeptides associees a des etats physiologiques et medicaux
AU784869B2 (en) Methods to identify evolutionarily significant changes in polynucleotide and polypeptide sequences in domesticated plants and animals
AU2007202866A1 (en) Methods to identify polynucleotide and polypeptide sequences which may be associated with physiological and medical conditions
EP2048249A1 (fr) Procédés pour identifier des séquences de polynucléotides et polypeptides pouvant être associées avec des états physiologiques et médicaux
Fearnley et al. Ultrafast, alignment-free detection of repeat expansions in next-generation DNA and RNA sequencing data
Pannecoucke EVALUATION OF THE INVOLVEMENT OF NOTCH1 VARIANTS IN CAROTID AND VERTEBRAL ARTERY DISSECTION
AU2003298556A8 (en) Functional sites
WO2004031397A2 (fr) Detection de goulots d'etranglements dans l'evolution par sequençage d'adn en tant que methode de decouverte de genes de valeur
Hill et al. Chromosome Substitution Strains

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AL AM AT AU AZ BA BB BG BR BY CA CH CN CR CU CZ DE DK DM 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 ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW SD SL SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
AK Designated states

Kind code of ref document: C2

Designated state(s): AE AL AM AT AU AZ BA BB BG BR BY CA CH CN CR CU CZ DE DK DM 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 ZA ZW

AL Designated countries for regional patents

Kind code of ref document: C2

Designated state(s): GH GM KE LS MW SD SL SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

COP Corrected version of pamphlet

Free format text: PAGES 2/28-3/28 AND 28/28, DRAWINGS, REPLACED BY NEW PAGES 2/28-3/28 AND 28/28; DUE TO LATE TRANSMITTAL BY THE RECEIVING OFFICE

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase