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Typically, the target data records are stored as a bit pattern in an array of magnetic domains on a magnetizable medium or as an array of charge states or transistor gate states, such chat co room 221576 an array of cells in a DRAM device e. In one embodiment, the invention provides such storage devices, and computer systems built therewith, comprising a bit pattern encoding a protein expression fingerprint record comprising unique identifiers for at least 10 target data records cross-tabulated with target source.
When the target is a peptide or nucleic acid, the invention preferably provides a method for identifying related peptide or nucleic acid sequences, comprising performing a computerized comparison between a peptide or nucleic acid sequence assay record stored in or retrieved from a computer storage device or database and at least one other sequence.SWIM SUITS FOR OLDER WOMEN
The comparison can include a sequence analysis or comparison algorithm or computer program embodiment thereof e, chat co room 221576. The invention also provides a network, comprising a plurality of computing devices linked via a data link, such as an Ethernet cable coax or lOBaseTtelephone line, ISDN line, wireless network, optical fiber, or other suitable signal transmission medium, whereby at least one network device e.
The invention also provides a method for transmitting assay data that includes generating an electronic signal on an electronic communications device, such as a modem, ISDN terminal adapter, DSL, cable modem, ATM switch, or the like, wherein the signal includes in native or encrypted format a chat co room 221576 pattern encoding data from an assay or a database comprising a plurality of assay results obtained by the method of the invention.
In a preferred embodiment, the invention provides a computer system for comparing a query target to a database containing an array of data structures, such as an assay result obtained by the method of the invention, and ranking database targets based on the degree of identity and gap weight to the target data. Execution of the computer program results in the central processor retrieving the assay data from the data file, which comprises a binary description of an assay result.
The target data or record and the computer program can be transferred to secondary memory, which is typically random access memory e.
Targets are ranked according to the degree daddy and dating site correspondence between a selected assay characteristic e.
For example, a central processor can be a conventional computer e. The invention also preferably provides the use of a computer system, such as that described above, which comprises: 1 a computer; 2 a stored bit pattern encoding a collection of peptide sequence specificity records obtained by the methods of the invention, which may be stored in the computer; 3 a comparison target, such as a query target; and 4 a program for alignment and comparison, typically with rank-ordering of comparison results on the basis of computed similarity values.
Prostate cancer proteins of the present invention may be classified as secreted proteins, transmembrane proteins or intracellular proteins. In one embodiment, the prostate cancer protein is an intracellular protein. Intracellular proteins are involved in all aspects of cellular function how do u find love replication including, e.
For example, many intracellular proteins have enzymatic activity such as protein kinase activity, protein phosphatase activity, protease activity, nucleotide cyclase activity, polymerase activity and the like. Intracellular proteins also serve as docking proteins that are involved in organizing complexes of proteins, or targeting proteins to various subcellular localizations, and are involved in maintaining the structural integrity of organelles.
An increasingly appreciated concept in characterizing proteins is the presence in the proteins of one or more motifs for which defined functions have been attributed. In addition to the highly conserved sequences found in chat co room 221576 enzymatic domain chat co room 221576 proteins, highly conserved sequences have been identified in proteins that are involved in protein-protein interaction.
For example, Src-homology-2 SH2 domains bind tyrosine-phosphorylated targets in a sequence dependent manner. PTB domains, which are distinct from SH2 domains, also bind tyrosine phosphorylated targets.
SH3 domains bind to proline-rich targets. In addition, PH domains, tetratricopeptide repeats and WD domains to name only a few, have been shown to mediate protein-protein interactions. Some of these may also be involved in binding to phospholipids or other second messengers. One useful database is Pfam protein familieswhich is a large collection of multiple sequence alignments and hidden Markov models covering many common protein domains.
Versions chat co room 221576 available via the internet from Washington University in St. In another embodiment, the prostate cancer sequences are transmembrane proteins. Transmembrane proteins are molecules that how long jade jed been dating a phospholipid bilayer of a cell. They may have an intracellular domain, an extracellular domain, or both.
The intracellular domains of such proteins may have a number of functions including those already described for intracellular proteins. Frequently the intracellular domain of transmembrane proteins serves both roles. For example certain receptor tyrosine kinases have both protein kinase activity and SH2 domains.
In addition, autophosphorylation of tyrosines on the receptor molecule itself, creates binding sites for additional SH2 domain containing proteins. Transmembrane proteins may contain from one to many transmembrane domains. However, various other chat co room 221576 including channels and adenylyl cyclases contain numerous transmembrane domains.
Many important cell surface receptors such as G protein coupled receptors GPCRs are classified as "seven transmembrane domain" proteins, as they contain 7 membrane spanning regions. Characteristics of transmembrane domains include approximately 20 consecutive hydrophobic amino acids that may be followed by charged amino acids. Therefore, upon analysis of the amino acid sequence of a particular protein, the localization and number of transmembrane domains within the protein may be predicted see, e.
Important transmembrane protein receptors include, but are not limited to the insulin receptor, insulin-like growth factor receptor, human growth hormone receptor, glucose transporters, transferrin receptor, epidermal growth factor receptor, low density lipoprotein receptor, epidermal growth factor receptor, leptin receptor, interleukin receptors, e.
IL-1 receptor, IL-2 receptor. The extracellular domains of transmembrane proteins are diverse; however, conserved motifs are found repeatedly among various extracellular domains. Many extracellular domains are involved in binding to other molecules. In one aspect, extracellular domains are found on receptors. Factors that bind the receptor domain include circulating ligands, which may be peptides, proteins, chat co room 221576, or small molecules such as chat co room 221576 and the like.
Other factors include cytokines, mitogenic factors, neurotrophic factors and the like. Extracellular domains also bind to cell-associated molecules. In this respect, they mediate cell-cell interactions. Cell- associated ligands can be tethered to the cell, e. Extracellular domains also associate with the extracellular matrix and contribute to the maintenance of the cell structure.
Prostate cancer proteins that are transmembrane are particularly preferred in the present invention as they are readily accessible targets chat co room 221576 immunotherapeutics, as are described herein. In addition, as outlined below, transmembrane proteins can be also useful in imaging modalities. Antibodies may be used to label such readily accessible proteins in situ. Alternatively, antibodies can also label intracellular proteins, in which case samples are typically permeablized to provide access to intracellular proteins.
It will also be appreciated by those in the art that a transmembrane protein can be made soluble by removing transmembrane sequences, e. Furthermore, transmembrane proteins that have been made soluble can be made to be secreted through recombinant means by adding an appropriate signal sequence.
In another embodiment, the prostate cancer proteins are secreted proteins; the secretion of which can be either constitutive or regulated. These proteins have a signal peptide or chat co room 221576 sequence that targets the molecule to the secretory pathway.
Secreted proteins are involved in numerous physiological events; by virtue of their circulating nature, they serve to transmit signals to various other cell types. The secreted protein may function in an autocrine manner acting on the cell that secreted the factora paracrine manner acting on cells in close proximity to the cell that secreted the factor or an endocrine manner acting on cells at a distance. Thus secreted molecules find use in modulating or altering numerous aspects of physiology.
Prostate cancer proteins that are secreted proteins are particularly preferred in the present invention as they serve as good targets for diagnostic markers, e. Typically, linked sequences on a mRNA are found on the same molecule. The prostate cancer nucleic acid sequences of the invention, e. Accordingly, as will be appreciated by those in the art, using the sequences provided herein, extended sequences, in either direction, of the prostate cancer genes can be obtained, using techniques well known in the art for cloning either longer sequences or the full length sequences; see Ausubel, et al, supra.
Much can be done by informatics and many sequences can be clustered to include multiple sequences corresponding to a single gene, e. Once the prostate cancer nucleic acid is identified, it can be cloned and, if necessary, its constituent parts recombined to form the entire prostate cancer nucleic acid coding regions or the entire mRNA sequence.
Once isolated from its natural source, e. It can also be used as a "precursor" nucleic acid to make modified or variant prostate cancer nucleic acids and proteins. The prostate cancer nucleic acids of easy mums dating review present invention are used in several ways. In a first embodiment, nucleic acid probes to the prostate cancer nucleic acids are made and attached to biochips to be used in screening and diagnostic methods, as outlined below, or for administration, e.
Alternatively, the prostate cancer nucleic acids that include coding regions of prostate cancer proteins can be put into expression vectors for the expression of prostate cancer proteins, again for screening purposes or for administration to a patient. The nucleic acid probes attached to the biochip are designed to be substantially complementary to the prostate cancer nucleic acids, i.
As outlined below, this complementarity need not be perfect; there may be any number of base pair mismatches which will interfere with hybridization between the target sequence and the single stranded nucleic acids of the present invention.
However, if the number of mutations is so great that no hybridization can occur under even the least stringent of hybridization conditions, the sequence is not a complementary target sequence.
Thus, chat co room 221576 "substantially complementary" herein is meant that the probes are sufficiently complementary to the target sequences to hybridize under normal reaction conditions, particularly high stringency conditions, as outlined herein. A nucleic acid probe is generally single stranded but can be partially single and partially double stranded.
The strandedness of the probe is dictated by the structure, composition, and properties of the target sequence. In general, the nucleic acid probes range from about 8 to about bases long, with from about 10 to about 80 bases being preferred, and from about 30 to about 50 bases being particularly preferred. That is, generally whole genes are not used. In some embodiments, much longer nucleic acids can be used, up to hundreds of bases. In a preferred embodiment, more than one probe per sequence is used, with either overlapping probes or probes to different sections of the target being used.
That is, two, three, four or more probes, with three being preferred, are used to build in a redundancy for a particular target. The probes can be overlapping i. In some cases, PCR primers may be used to amplify signal for higher sensitivity. As will be appreciated by those in the art, nucleic acids can be attached or immobilized to a solid support in a wide variety of ways. By "immobilized" and grammatical equivalents herein is meant the association or binding between the nucleic acid probe and the solid support is sufficient to be stable under the conditions of binding, washing, analysis, and removal as outlined chat co room 221576.
The binding can typically be covalent or non-covalent.SINGLE CHAT ROOMS
By "non- covalent binding" and grammatical equivalents herein is meant one or more of electrostatic, hydrophilic, and hydrophobic interactions. Included in non-covalent binding is the covalent attachment of a molecule, such as, streptavidin to the support and the non-covalent binding of the biotinylated probe to the streptavidin. By "covalent binding" and grammatical equivalents herein is meant that the two moieties, the solid support and the probe, are attached by at least one bond, including sigma bonds, pi chat co room 221576 and coordination bonds.
Covalent bonds can be formed directly between the probe and the solid support or can be formed by a cross linker or by inclusion of a specific reactive group on either the solid support or the probe or both molecules.
Immobilization may also involve a combination of covalent and non-covalent interactions. In general, the probes are attached to the biochip in a wide variety of ways, as will be appreciated by those in the art.
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As described herein, the nucleic acids can either be synthesized first, with subsequent attachment to the biochip, or can be directly synthesized on the biochip. The biochip comprises a suitable solid substrate.
By "substrate" or "solid support" or other grammatical equivalents herein is meant a material that can be modified to contain discrete individual sites appropriate for the attachment or association of the nucleic acid probes and is amenable to at least one detection method.
As single chat rooms be appreciated by those in the art, the number of possible substrates are very large, and include, but are not limited to, glass and modified or functionalized glass, plastics including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, TeflonJ, etc.
In general, the substrates allow optical detection and do not appreciably fluoresce. Application Serial No. Generally the substrate is planar, although as will be appreciated by those in the art, other configurations of substrates may be used as well. For example, the probes may be placed on the inside surface of a tube, for flow-through sample analysis chat co room 221576 minimize sample volume.
Similarly, the substrate may be flexible, such as a flexible foam, including closed cell foams made of particular plastics. In a preferred embodiment, the surface of the biochip and the probe may be derivatized with chemical functional groups for subsequent attachment of the chat co room 221576.
Using these functional groups, the probes can be attached using functional groups on the probes. For example, nucleic acids containing amino groups can be attached to surfaces comprising amino groups, e. In addition, in some cases, additional linkers, such as alkyl groups including substituted and heteroalkyl groups may be used.
In this embodiment, oligonucleotides are synthesized as is known in the art, and then attached to the surface of the solid support. As will be appreciated by those skilled in the art, either the 5' or 3' terminus may be attached to the solid support, or attachment may be via an internal nucleoside. In another embodiment, the immobilization to the solid support may be very strong, yet non-covalent.
For example, biotinylated oligonucleotides can be made, which bind to surfaces covalently coated with streptavidin, resulting in attachment. Alternatively, the oligonucleotides may be synthesized on the surface, as is known in the art. For example, photoactivation techniques utilizing photopolymerization compounds and techniques are used.
Often, amplification-based assays are performed to measure the expression level of prostate cancer-associated sequences. These assays are typically performed in conjunction with reverse transcription. In such assays, a prostate cancer-associated nucleic acid sequence acts as a template in an amplification reaction e. In a quantitative amplification, the amount of amplification product will be proportional to the amount of template in the original sample. Comparison to appropriate controls provides a measure of the amount of prostate cancer-associated RNA.
Methods of quantitative amplification are well known to those of skill in the art. Detailed protocols for quantitative PCR are provided, e. In some embodiments, a TaqMan based assay is used chat co room 221576 measure expression. TaqMan based assays use a fluorogenic oligonucleotide probe that contains a 5' fluorescent dye and a 3' quenching agent.
Chat co room 221576 probe hybridizes to a PCR product, but cannot itself be extended due to a blocking agent at the 3' end. When the PCR product is amplified in subsequent cycles, the chat co room 221576 nuclease activity of the polymerase, e. This cleavage separates the 5' fluorescent dye and the 3' quenching agent, thereby resulting in an increase in fluorescence as a function of chat co room 221576 see, e.
USAself-sustained sequence replication Guatelli et al, Proc. In a preferred embodiment, meet big booty cancer nucleic acids, e. Expression vectors and recombinant DNA technology are well known to those of skill in the art see, e. The expression vectors may be either self-replicating extrachromosomal vectors or vectors which integrate into a host genome.
Generally, these expression vectors include transcriptional and translational regulatory nucleic acid operably linked to the nucleic acid encoding the prostate cancer protein.
The term "control sequences" refers to DNA sequences used for the expression of an operably linked coding sequence in a particular host organism, chat co room 221576. Control sequences that are suitable for prokaryotes, e. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers. Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a dating girl with guy or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.
Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is typically accomplished by ligation at convenient restriction sites.
If such sites do not exist, synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice. Transcriptional and translational regulatory nucleic acid will generally be appropriate to the host cell used to express the prostate cancer protein. Numerous types of appropriate expression vectors, and suitable regulatory sequences are known in the art for a variety of host cells.free mobile chat room asia girls
In general, transcriptional and translational regulatory sequences may include, but are not limited to, promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences.
In a preferred embodiment, the regulatory sequences include a promoter and transcriptional start and stop sequences.
Promoter sequences find travel friends app either constitutive or inducible promoters.
The promoters may be either naturally occurring promoters or hybrid promoters. Hybrid promoters, which combine elements of more than one promoter, are also known in the art, and are useful in the present invention. In addition, an expression vector may comprise additional elements.
For example, the expression vector may have two replication systems, thus allowing it to be maintained in two organisms, e. Furthermore, for integrating expression vectors, the expression vector contains at least one sequence homologous to the host cell genome, and preferably two homologous sequences which flank the expression construct.
The integrating vector may be directed to a specific locus in the host cell by selecting the appropriate homologous sequence for inclusion in the vector. Constructs for integrating vectors are well known in the art e.
In addition, in a preferred embodiment, the expression vector contains a selectable marker gene to allow the selection of transformed host cells. Selection genes are well known in the art and will vary with the host cell used. The prostate cancer proteins of the present invention are produced by culturing a host cell transformed with an expression vector containing nucleic acid encoding a prostate cancer protein, under the appropriate conditions to induce or cause expression of the prostate cancer protein.
Conditions appropriate for prostate cancer protein expression will vary with the choice of the expression vector and the host cell, chat co room 221576 will be easily ascertained by one skilled in the chat co room 221576 through routine experimentation or optimization.
For example, the use of constitutive promoters in the expression vector will require optimizing the growth and proliferation of the host cell, while the use of an inducible promoter requires the appropriate growth conditions for induction. In addition, in some embodiments, the timing of the harvest is important. For example, the baculo viral systems used in insect cell expression are lytic viruses, and thus harvest time selection can be crucial for product yield.
Appropriate host cells include yeast, bacteria, archaebacteria, fungi, chat co room 221576, and insect and animal cells, including mammalian cells. Of particular interest are Saccharomyces cerevisiae and other yeasts, E.
In a preferred embodiment, the prostate cancer proteins are expressed in mammalian cells. Mammalian expression systems are also known in the art, and include retroviral and adenoviral systems. Of particular use as mammalian promoters are the promoters from mammalian viral genes, since the viral genes are often highly expressed and have a broad host range.
Examples include the S V40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter, herpes simplex virus promoter, and the CMV promoter see, e. Typically, transcription termination and polyadenylation sequences recognized by mammalian cells are regulatory regions located 3' to the translation stop codon and thus, together with the promoter elements, flank the coding sequence.
Examples of transcription terminator and polyadenlyation signals include those derived form SV The methods of introducing exogenous nucleic acid into mammalian hosts, as well as other hosts, is well known in the art, and will vary with the host cell used. Techniques include dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated chat co room 221576, protoplast fusion, electroporation, viral infection, encapsulation of the polynucleotide s chat co room 221576 liposomes, and direct microinjection of the DNA into nuclei.
In a preferred embodiment, prostate cancer proteins are expressed in bacterial systems. Bacterial expression systems are well known in the art. Promoters from bacteriophage may also be used and are known in the art.
In addition, synthetic promoters and hybrid chat co room 221576 are also useful; e. Furthermore, a bacterial promoter can include naturally occurring promoters of non-bacterial origin that have the ability to bind bacterial RNA polymerase and initiate transcription. In addition to a functioning promoter sequence, an efficient ribosome binding site is desirable. The expression vector may also include a signal peptide sequence that provides for secretion of the prostate cancer protein in bacteria.
The protein is either secreted into the growth media gram-positive bacteria or into the periplasmic space, located between the inner and outer membrane of the cell gram-negative bacteria. The bacterial expression vector may also include a selectable marker gene to allow for the selection of bacterial strains that have been transformed.
Suitable selection genes include genes which render the bacteria resistant to drugs such as ampicillin, chloramphenicol, erythromycin, kanamycin, neomycin and tetracycline. Selectable markers also include biosynthetic genes, such as those in the histidine, tryptophan and leucine biosynthetic pathways.
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