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1. 6For both caraway forms, accumulation of limonene and carvone in the fruits is a developmentally regulated process. Generally, a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid.
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5. 3 For example, a polynucleotide sequence in a library can be a polynucleotide that represents an mRNA, polypeptide, or other gene product encoded by the polynucleotide, that is either overexpressed or underexpressed in a cancerous cell affected by cancer relative to a normal i.
6. 7 Exemplary uses of arrays are further described in, for example, Pappalarado et al. In some embodiments, the methods comprise:
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In other specific embodiments detection of expression of the gene is by detecting a level of a polypeptide in a test sample. The invention finds use in the prevention, treatment, detection or research into any cancer, including prostrate, pancreas, colon, brain, lung, breast, bone, skin cancers. These polynucleotides, polypeptides and antibodies are thus useful in a variety of diagnostic, therapeutic, and drug discovery methods. Another dilemma you could learn with no cost software, is always that the creator might or might not retain it up to date. The polynucleotides can be introduced into suitable host cells using a variety of techniques available in the art, such as transferrin polycation-mediated DNA transfer, transfection with naked or encapsulated nucleic acids, liposome-mediated DNA transfer, intracellular transportation of DNA-coated latex beads, protoplast fusion, viral infection, electroporation, gene gun, calcium phosphate-mediated transfection, and the like.
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In other embodiments, the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e. In yet other embodiments, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, e.
As with the instructions, this means for obtaining the instructions is recorded on a suitable substrate. In general, a library of polynucleotides is a collection of sequence information, which information is provided in either biochemical form e.
The sequence information of the polynucleotides can be used in a variety of ways, e. For example, in the instant case, the sequences of polynucleotides and polypeptides corresponding to genes differentially expressed in cancer, as well as the nucleic acid and amino acid sequences of the genes themselves, can be provided in electronic form in a computer database.
In general, a disease marker is a representation of a gene product that is present in all cells affected by disease either at an increased or decreased level relative to a normal cell e.
For example, a polynucleotide sequence in a library can be a polynucleotide that represents an mRNA, polypeptide, or other gene product encoded by the polynucleotide, that is either overexpressed or underexpressed in a cancerous cell affected by cancer relative to a normal i.
The nucleotide sequence information of the library can be embodied in any suitable form, e. For example, a library of sequence information embodied in electronic form comprises an accessible computer data file or, in biochemical form, a collection of nucleic acid molecules that contains the representative nucleotide sequences of genes that are differentially expressed e.
Other combinations and comparisons of cells affected by various diseases or stages of disease will be readily apparent to the ordinarily skilled artisan. Biochemical embodiments of the library include a collection of nucleic acids that have the sequences of the genes in the library, where the nucleic acids can correspond to the entire gene in the library or to a fragment thereof, as described in greater detail below.
The polynucleotide libraries of the subject invention generally comprise sequence information of a plurality of polynucleotide sequences, where at least one of the polynucleotides has a sequence of any of sequence described herein.
By plurality is meant at least 2, usually at least 3 and can include up to all of the sequences described herein. The length and number of polynucleotides in the library will vary with the nature of the library, e.
Where the library is an electronic library, the nucleic acid sequence information can be present in a variety of media. Such a manufacture provides the genome sequence or a subset thereof in a form that can be examined by means not directly applicable to the sequence as it exists in a nucleic acid.
For example, the nucleotide sequence of the present invention, e. Such media include, but are not limited to: One of skill in the art can readily appreciate how any of the presently known computer readable mediums can be used to create a manufacture comprising a recording of the present sequence information.
Any convenient data storage structure can be chosen, based on the means used to access the stored information. A variety of data processor programs and formats can be used for storage, e.
By providing the nucleotide sequence in computer readable form, the information can be accessed for a variety of purposes. Computer software to access sequence information e.
The minimum hardware of the computer-based systems of the present invention comprises a central processing unit CPU , input means, output means, and data storage means. A skilled artisan can readily appreciate that any one of the currently available computer-based system are suitable for use in the present invention.
The data storage means can comprise any manufacture comprising a recording of the present sequence information as described above, or a memory access means that can access such a manufacture.
Search means can be used to identify fragments or regions of the genome that match a particular target sequence or target motif. A variety of known algorithms are publicly known and commercially available, e.
A variety of means for comparing nucleic acids or polypeptides may be used to compare accomplish a sequence comparison e. A skilled artisan can readily recognize that any one of the publicly available homology search programs can be used to search the computer based systems of the present invention to compare of target sequences and motifs.
Computer programs to analyze expression levels in a sample and in controls are also known in the art. There are a variety of target motifs known in the art.
Protein target motifs include, but are not limited to, enzyme active sites and signal sequences, kinase domains, receptor binding domains, SH2 domains, SH3 domains, phosphorylation sites, protein interaction domains, transmembrane domains, etc.
Nucleic acid target motifs include, but are not limited to, hairpin structures, promoter sequences and other expression elements such as binding sites for transcription factors.
A variety of structural formats for the input and output means can be used to input and output the information in the computer-based systems of the present invention. One format for an output means ranks the relative expression levels of different polynucleotides.
Such presentation provides a skilled artisan with a ranking of relative expression levels to determine a gene expression profile. A gene expression profile can be generated from, for example, a cDNA library prepared from mRNA isolated from a test cell suspected of being cancerous or pre-cancerous, comparing the sequences or partial sequences of the clones against the sequences in an electronic database, where the sequences of the electronic database represent genes differentially expressed in a cancerous cell, e.
The number of clones having a sequence that has substantial similarity to a sequence that represents a gene differentially expressed in a cancerous cell is then determined, and the number of clones corresponding to each of such genes is determined.
An increased number of clones that correspond to differentially expressed gene is present in the cDNA library of the test cell relative to, for example, the number of clones expected in a cDNA of a normal cell indicates that the test cell is cancerous.
The biochemical libraries can take a variety of forms, e. Of particular interest are nucleic acid arrays in which one or more of the genes described herein is represented by a sequence on the array.
By array is meant an article of manufacture that has at least a substrate with at least two distinct nucleic acid targets on one of its surfaces, where the number of distinct nucleic acids can be considerably higher, typically being at least 10 nt, usually at least 20 nt and often at least 25 nt.
A variety of different array formats have been developed and are known to those of skill in the art. The arrays of the subject invention find use in a variety of applications, including gene expression analysis, drug screening, mutation analysis and the like, as disclosed in the above-listed exemplary patent documents.
In addition to the above nucleic acid libraries, analogous libraries of polypeptides are also provided, where the polypeptides of the library will represent at least a portion of the polypeptides encoded by a gene corresponding to a sequence described herein.
The present invention provides methods of using the polynucleotides described herein in, for example, diagnosis of cancer and classification of cancer cells according to expression profiles.
In specific non-limiting embodiments, the methods are useful for detecting cancer cells, facilitating diagnosis of cancer and the severity of a cancer e. The detection methods of the invention can be conducted in vitro or in vivo, on isolated cells, or in whole tissues or a bodily fluid, e.
In general, methods of the invention involving detection of a gene product e. The probe and sample suspected of having the gene product of interest are contacted under conditions suitable for binding of the probe to the gene product.
For example, contacting is generally for a time sufficient to allow binding of the probe to the gene product e. Suitable conditions for probe-target gene product binding can be readily determined using controls and other techniques available and known to one of ordinary skill in the art.
In this embodiment, the probe can be an antibody or other polypeptide, peptide, or molecule e. The detection methods can be provided as part of a kit. Procedures using these kits can be performed by clinical laboratories, experimental laboratories, medical practitioners, or private individuals.
The kits of the invention for detecting a polypeptide encoded by a polynucleotide that is differentially expressed in a cancer cell comprise a moiety that specifically binds the polypeptide, which may be a specific antibody.
The kits of the invention for detecting a polynucleotide that is differentially expressed in a cancer cell comprise a moiety that specifically hybridizes to such a polynucleotide.
The kit may optionally provide additional components that are useful in the procedure, including, but not limited to, buffers, developing reagents, labels, reacting surfaces, means for detection, control samples, standards, instructions, and interpretive information.
In some embodiments, methods are provided for a detecting cancer cell by detecting in a cell, a polypeptide encoded by a gene differentially expressed in a cancer cell. Any of a variety of known methods can be used for detection, including, but not limited to, immunoassay, using an antibody specific for the encoded polypeptide, e.
For example, an immunofluorescence assay can be easily performed on cells without first isolating the encoded polypeptide. The cells are first fixed onto a solid support, such as a microscope slide or microtiter well.
This fixing step can permeabilize the cell membrane. The permeabilization of the cell membrane permits the polypeptide-specific probe e. Alternatively, where the polypeptide is secreted or membrane-bound, or is otherwise accessible at the cell-surface e.
Next, the fixed cells are exposed to an antibody specific for the encoded polypeptide. To increase the sensitivity of the assay, the fixed cells may be further exposed to a second antibody, which is labeled and binds to the first antibody, which is specific for the encoded polypeptide.
Typically, the secondary antibody is detectably labeled, e. The cells which express the encoded polypeptide will be fluorescently labeled and easily visualized under the microscope. See, for example, Hashido et al.
As will be readily apparent to the ordinarily skilled artisan upon reading the present specification, the detection methods and other methods described herein can be varied. Such variations are within the intended scope of the invention.
For example, in the above detection scheme, the probe for use in detection can be immobilized on a solid support, and the test sample contacted with the immobilized probe. Binding of the test sample to the probe can then be detected in a variety of ways, e.
In this embodiment, the probe can be a an antibody or other polypeptide, peptide, or molecule e. The methods generally comprise: The level of antibody binding either qualitative or quantitative indicates the cancerous state of the cell.
For example, where the differentially expressed gene is increased in cancerous cells, detection of an increased level of antibody binding to the test sample relative to antibody binding level associated with a normal cell indicates that the test cell is cancerous.
Suitable controls include a sample known not to contain the encoded polypeptide; and a sample contacted with an antibody not specific for the encoded polypeptide, e. A variety of methods to detect specific antibody-antigen interactions are known in the art and can be used in the method, including, but not limited to, standard immunohistological methods, immunoprecipitation, an enzyme immunoassay, and a radioimmunoassay.
In general, the specific antibody will be detectably labeled, either directly or indirectly. Direct labels include radioisotopes; enzymes whose products are detectable e.
The antibody may be attached coupled to an insoluble support, such as a polystyrene plate or a bead. The biological sample may be brought into contact with and immobilized on a solid support or carrier, such as nitrocellulose, that is capable of immobilizing cells, cell particles, or soluble proteins.
The support may then be washed with suitable buffers, followed by contacting with a detectably-labeled first specific antibody. Detection methods are known in the art and will be chosen as appropriate to the signal emitted by the detectable label.
Detection is generally accomplished in comparison to suitable controls, and to appropriate standards. In some embodiments, the methods are adapted for use in vivo, e. In these embodiments, a detectably-labeled moiety, e.
In this manner, cancer cells are differentially labeled. In some embodiments, methods are provided for detecting a cancer cell by detecting expression in the cell of a transcript or that is differentially expressed in a cancer cell.
Any of a variety of known methods can be used for detection, including, but not limited to, detection of a transcript by hybridization with a polynucleotide that hybridizes to a polynucleotide that is differentially expressed in a cancer cell; detection of a transcript by a polymerase chain reaction using specific oligonucleotide primers; in situ hybridization of a cell using as a probe a polynucleotide that hybridizes to a gene that is differentially expressed in a cancer cell and the like.
In many embodiments, the levels of a subject gene product are measured. By measured is meant qualitatively or quantitatively estimating the level of the gene product in a first biological sample either directly e.
In many embodiments the second control biological sample is obtained from an individual not having not having cancer. As will be appreciated in the art, once a standard control level of gene expression is known, it can be used repeatedly as a standard for comparison.
Other control samples include samples of cancerous tissue. In some embodiments, the methods comprise: Detection of differential hybridization, when compared to a suitable control, is an indication of the presence in the sample of a polynucleotide that is differentially expressed in a cancer cell.
Appropriate controls include, for example, a sample that is known not to contain a polynucleotide that is differentially expressed in a cancer cell. Conditions that allow hybridization are known in the art, and have been described in more detail above.
A variety of labels and labeling methods for polynucleotides are known in the art and can be used in the assay methods of the invention. Specific hybridization can be determined by comparison to appropriate controls.
Additional disclosure about preferred regions of the disclosed polynucleotide sequences is found in the Examples. A probe that hybridizes specifically to a polynucleotide disclosed herein should provide a detection signal at least 2-, 5-, , or fold higher than the background hybridization provided with other unrelated sequences.
As will be readily appreciated by the ordinarily skilled artisan, the probe can be detectably labeled and contacted with, for example, an array comprising immobilized polynucleotides obtained from a test sample e.
Alternatively, the probe can be immobilized on an array and the test sample detectably labeled. These and other variations of the methods of the invention are well within the skill in the art and are within the scope of the invention.
Labeled nucleic acid probes may be used to detect expression of a gene corresponding to the provided polynucleotide. In Northern blots, mRNA is separated electrophoretically and contacted with a probe.
A probe is detected as hybridizing to an mRNA species of a particular size. The amount of hybridization can be quantitated to determine relative amounts of expression, for example under a particular condition.
Probes are used for in situ hybridization to cells to detect expression. Probes can also be used in vivo for diagnostic detection of hybridizing sequences. Probes are typically labeled with a radioactive isotope.
Other types of detectable labels can be used such as chromophores, fluorophores, and enzymes. PCR is another means for detecting small amounts of target nucleic acids, methods for which may be found in Sambrook, et al.
A detectable label may be included in the amplification reaction. Suitable detectable labels include fluorochromes, e. The label may be a two stage system, where the polynucleotides is conjugated to biotin, haptens, etc.
The label may be conjugated to one or both of the primers. Alternatively, the pool of nucleotides used in the amplification is labeled, so as to incorporate the label into the amplification product.
Polynucleotide arrays provide a high throughput technique that can assay a large number of polynucleotides or polypeptides in a sample. This technology can be used as a tool to test for differential expression.
A variety of methods of producing arrays, as well as variations of these methods, are known in the art and contemplated for use in the invention. For example, arrays can be created by spotting polynucleotide probes onto a substrate e.
The probes can be bound to the substrate by either covalent bonds or by non-specific interactions, such as hydrophobic interactions. Samples of polynucleotides can be detectably labeled e.
Double stranded polynucleotides, comprising the labeled sample polynucleotides bound to probe polynucleotides, can be detected once the unbound portion of the sample is washed away. Alternatively, the polynucleotides of the test sample can be immobilized on the array, and the probes detectably labeled.
Techniques for constructing arrays and methods of using these arrays are described in, for example, Schena et al. In other embodiments, the probe is immobilized on the array and not detectably labeled.
Arrays can be used, for example, to examine differential expression of genes and can be used to determine gene function. For example, arrays can be used to detect differential expression of a gene corresponding to a polynucleotide described herein, where expression is compared between a test cell and control cell e.
For example, high expression of a particular message in a cancer cell, which is not observed in a corresponding normal cell, can indicate a cancer specific gene product. Exemplary uses of arrays are further described in, for example, Pappalarado et al.
Furthermore, many variations on methods of detection using arrays are well within the skill in the art and within the scope of the present invention. For example, rather than immobilizing the probe to a solid support, the test sample can be immobilized on a solid support which is then contacted with the probe.
The polynucleotides described herein, as well as their gene products and corresponding genes and gene products, are of particular interest as genetic or biochemical markers e. For example, the level of expression of certain polynucleotides can be indicative of a poorer prognosis, and therefore warrant more aggressive chemo- or radio-therapy for a patient or vice versa.
The correlation of novel surrogate tumor specific features with response to treatment and outcome in patients can define prognostic indicators that allow the design of tailored therapy based on the molecular profile of the tumor.
These therapies include antibody targeting, antagonists e. Determining expression of certain polynucleotides and comparison of a patient’s profile with known expression in normal tissue and variants of the disease allows a determination of the best possible treatment for a patient, both in terms of specificity of treatment and in terms of comfort level of the patient.
Surrogate tumor markers, such as polynucleotide expression, can also be used to better classify, and thus diagnose and treat, different forms and disease states of cancer. Two classifications widely used in oncology that can benefit from identification of the expression levels of the genes corresponding to the polynucleotides described herein are staging of the cancerous disorder, and grading the nature of the cancerous tissue.
The polynucleotides that correspond to differentially expressed genes, as well as their encoded gene products, can be useful to monitor patients having or susceptible to cancer to detect potentially malignant events at a molecular level before they are detectable at a gross morphological level.
In addition, the polynucleotides described herein, as well as the genes corresponding to such polynucleotides, can be useful as therametrics, e. Furthermore, a polynucleotide identified as corresponding to a gene that is differentially expressed in, and thus is important for, one type of cancer can also have implications for development or risk of development of other types of cancer, e.
Thus, for example, expression of a polynucleotide corresponding to a gene that has clinical implications for cancer can also have clinical implications for metastatic breast cancer, colon cancer, or ovarian cancer, etc.
Staging is a process used by physicians to describe how advanced the cancerous state is in a patient. Staging assists the physician in determining a prognosis, planning treatment and evaluating the results of such treatment.
Generally, if a cancer is only detectable in the area of the primary lesion without having spread to any lymph nodes it is called Stage I. If it has spread only to the closest lymph nodes, it is called Stage II.
In Stage III, the cancer has generally spread to the lymph nodes in near proximity to the site of the primary lesion. Cancers that have spread to a distant part of the body, such as the liver, bone, brain or other site, are Stage IV, the most advanced stage.
The polynucleotides and corresponding genes and gene products described herein can facilitate fine-tuning of the staging process by identifying markers for the aggressiveness of a cancer, e.
Thus, a Stage II cancer with a polynucleotide signifying a high metastatic potential cancer can be used to change a borderline Stage II tumor to a Stage III tumor, justifying more aggressive therapy.
Conversely, the presence of a polynucleotide signifying a lower metastatic potential allows more conservative staging of a tumor. One type of breast cancer is ductal carcinoma in situ DCIS: DCIS is when the breast cancer cells are completely contained within the breast ducts the channels in the breast that carry milk to the nipple , and have not spread into the surrounding breast tissue.
This may also be referred to as non-invasive or intraductal cancer, as the cancer cells have not yet spread into the surrounding breast tissue and so usually have not spread into any other part of the body.
Lobular carcinoma in situ breast cancer LCIS means that cell changes are found in the lining of the lobules of the breast. It can be present in both breasts. It is also referred to as non-invasive cancer as it has not spread into the surrounding breast tissue.
Invasive breast cancer can be staged as follows: The lymph glands in the armpit are not affected and there are no signs that the cancer has spread elsewhere in the body; Stage 2 tumours: However, there are no signs that the cancer has spread further; Stage 3 tumours: The lymph glands are usually affected, but there are no signs that the cancer has spread beyond the breast or the lymph glands in the armpit; Stage 4 tumours: This is secondary breast cancer.
Grade is a term used to describe how closely a tumor resembles normal tissue of its same type. The microscopic appearance of a tumor is used to identify tumor grade based on parameters such as cell morphology, cellular organization, and other markers of differentiation.
As a general rule, the grade of a tumor corresponds to its rate of growth or aggressiveness, with undifferentiated or high-grade tumors generally being more aggressive than well-differentiated or low-grade tumors.
The polynucleotides of the Sequence Listing, and their corresponding genes and gene products, can be especially valuable in determining the grade of the tumor, as they not only can aid in determining the differentiation status of the cells of a tumor, they can also identify factors other than differentiation that are valuable in determining the aggressiveness of a tumor, such as metastatic potential.
Low grade means that the cancer cells look very like the normal cells. They are usually slowly growing and are less likely to spread. In high grade tumors the cells look very abnormal.
They are likely to grow more quickly and are more likely to spread. Assessment of proliferation of cells in tumor. The differential expression level of the polynucleotides described herein can facilitate assessment of the rate of proliferation of tumor cells, and thus provide an indicator of the aggressiveness of the rate of tumor growth.
For example, assessment of the relative expression levels of genes involved in cell cycle can provide an indication of cellular proliferation, and thus serve as a marker of proliferation. The polynucleotides corresponding to genes that exhibit the appropriate expression pattern can be used to detect cancer in a subject.
The expression of appropriate polynucleotides can be used in the diagnosis, prognosis and management of cancer. Detection of cancer can be determined using expression levels of any of these sequences alone or in combination with the levels of expression of other known cancer genes.
For example, development of cancer can be detected by examining the level of expression of a gene corresponding to a polynucleotides described herein to the levels of oncogenes e.
Thus expression of specific marker polynucleotides can be used to discriminate between normal and cancerous tissue, to discriminate between cancers with different cells of origin, to discriminate between cancers with different potential metastatic rates, etc.
For a review of other markers of cancer, see, e. The invention further provides methods for reducing growth of cancer cells. The present invention provides methods for treating cancer, generally comprising administering to an individual in need thereof a substance that reduces cancer cell growth, in an amount sufficient to reduce cancer cell growth and treat the cancer.
Whether a substance, or a specific amount of the substance, is effective in treating cancer can be assessed using any of a variety of known diagnostic assays for cancer, including, but not limited to, proctoscopy, rectal examination, biopsy, contrast radiographic studies, CAT scan, and detection of a tumor marker associated with cancer in the blood of the individual e.
The substance can be administered systemically or locally. Thus, in some embodiments, the substance is administered locally, and cancer growth is decreased at the site of administration.
Local administration may be useful in treating, e. A substance that reduces cancer cell growth can be targeted to a cancer cell. Thus, in some embodiments, the invention provides a method of delivering a drug to a cancer cell, comprising administering a drug-antibody complex to a subject, wherein the antibody is specific for a cancer-associated polypeptide, and the drug is one that reduces cancer cell growth, a variety of which are known in the art.
Targeting can be accomplished by coupling e. Methods of coupling a drug to an antibody are well known in the art and need not be elaborated upon herein. Differentially expressed genes can be analyzed for correlation with other differentially expressed genes in a single tumor type or across tumor types.
Genes that demonstrate consistent correlation in expression profile in a given cancer cell type e. Tumors can then be classified according to the expression profile of one or more genes selected from one or more groups.
The tumor of each patient in a pool of potential patients can be classified as described above. Patients having similarly classified tumors can then be selected for participation in an investigative or clinical trial of a cancer therapeutic where a homogeneous population is desired.
The tumor classification of a patient can also be used in assessing the efficacy of a cancer therapeutic in a heterogeneous patient population. In addition, therapy for a patient having a tumor of a given expression profile can then be selected accordingly.
In another embodiment, differentially expressed gene products e. The growth of cancer cells is naturally limited in part due to immune surveillance. Stimulation of the immune system using a particular tumor-specific antigen enhances the effect towards the tumor expressing the antigen.
An active vaccine comprising a polypeptide encoded by the cDNA of this invention would be appropriately administered to subjects having an alteration, e.
Polypeptide antigens are typically combined with an adjuvant as part of a vaccine composition. The vaccine is preferably administered first as a priming dose, and then again as a boosting dose, usually at least four weeks later.
Further boosting doses may be given to enhance the effect. The dose and its timing are usually determined by the person responsible for the treatment. The invention also encompasses the selection of a therapeutic regimen based upon the expression profile of differentially expressed genes in the patient’s tumor.
The expression patterns of the tumor are then compared to the expression patterns of tumors that respond to a selected therapy. Where the expression profiles of the test tumor cell and the expression profile of a tumor cell of known drug responsivity at least substantially match e.
The TEP is compared to a reference expression pattern REP , which is generated by detection of expression of the selected set of genes in a reference sample e.
The selected set of genes includes at least one of the genes of the invention, which genes correspond to the polynucleotide sequences described herein. Of particular interest is a selected set of genes that includes gene differentially expressed in the disease for which the test sample is to be screened.
The present invention also encompasses methods for identification of agents having the ability to modulate activity of a differentially expressed gene product, as well as methods for identifying a differentially expressed gene product as a therapeutic target for treatment of cancer.
Identification of compounds that modulate activity of a differentially expressed gene product can be accomplished using any of a variety of drug screening techniques. Such agents are candidates for development of cancer therapies.
Of particular interest are screening assays for agents that have tolerable toxicity for normal, non-cancerous human cells. Screening assays can be based upon any of a variety of techniques readily available and known to one of ordinary skill in the art.
In general, the screening assays involve contacting a cancerous cell with a candidate agent, and assessing the effect upon biological activity of a differentially expressed gene product.
The effect upon a biological activity can be detected by, for example, detection of expression of a gene product of a differentially expressed gene e. Alternatively or in addition, the effect of the candidate agent can be assessed by examining the effect of the candidate agent in a functional assay.
When crude extracts of caraway fruits from different stages of development were assayed for monoterpene synthase activity with GPP as the substrate, limonene was the only monoterpene detected by radio-GLC, with the exception of small amounts of geraniol, a product of phosphohydrolase activity.
Thus, the cyclization of GPP to limonene is the first step of carvone biosynthesis in caraway. The activity was operationally soluble confined to the , g supernatant , displayed a pH optimum of approximately 7.
After anion-exchange chromatography to remove the endogenous limonene present in the crude extract, GPP was found to be converted to B, Product of partially purified limonene synthase.
For further details, see Methods. The supernatant fraction of caraway fruit extracts possessed a very active trans -carveol dehydrogenase activity. Substantial ethanol dehydrogenase activity was detected at pH 8.
Dehydrogenase activity with various carveol isomers as substrates in 50 m m Gly buffer, pH The pattern of accumulation of the monoterpenes limonene and carvone in developing caraway fruits was investigated by extracting fruits of nine different developmental stages in hexane and by analyzing the extracts with GC.
The levels of fatty acids in these extracts both free and bound as triacylglycerols were also measured by quantifying the fatty acid methyl esters formed after hydrolysis and transesterification in methanolic KOH.
The patterns of accumulation of limonene, carvone, and fatty acids and the activities of the three enzymes assayed were similar for annual and biennial caraway.
Therefore, only data for annual caraway are shown. Young fruits contained very low levels of carvone and fatty acids, but high concentrations of limonene, up to 1.
From about 10 DAP, carvone content increased rapidly until about 15 DAP, at which time it became approximately the same as that of limonene. From about 15 DAP, the concentration of both monoterpenes followed a similar pattern until the end of the experiment at 35 DAP.
Figure 4 A shows clearly that the accumulation of monoterpenes is confined to the early stages of fruit development and that limonene accumulation precedes carvone accumulation by a period of 5 to 10 d.
Dotted lines in A indicate sigmoidal curves of the equation: The dotted line in B indicates the carvone accumulation rate, which was calculated by taking the 1st-order derivative of the sigmoidal-curve fit to the carvone content data as shown in A.
Data for A were obtained from pooled samples of 0. Data for B were obtained from pooled samples of 1. Enzyme assays were carried out in triplicate limonene synthase or in duplicate other activities under linear conditions.
Error bars indicate se. The accumulation of fatty acids occurred late in fruit development, beginning at 15 to 25 DAP, and increased steadily until the end of the experiment to about 1 to 1.
Experiments to determine the rate of monoterpene and fatty acid biosynthesis by measuring the incorporation of [U- 14 C]Suc into pentane-soluble compounds showed similar trends. In the youngest stages 5—7 DAP , the majority of label was incorporated into limonene, with less incorporation into carvone and little or no incorporation into fatty acids.
No radiolabel was observed in the monoterpene intermediate trans -carveol at any developmental stage. The rate of radiolabel incorporation into limonene declined rapidly with development and was not detectable after 15 DAP.
The incorporation of [U- 14 C]Suc into carvone increased as the rate of incorporation into limonene declined, but was also undetectable after 15 DAP. From this period onward, the only pentane-soluble compounds to incorporate [U- 14 C]Suc were the fatty acids.
The level of activity of the three enzymes of monoterpene biosynthesis varied considerably over the period of fruit development Fig. In the youngest stages, both limonene synthase and trans -carveol dehydrogenase were active but limonenehydroxylase was not; however, the maximum in all three activities occurred at about 15 DAP.
From this stage, the activities of all three enzymes declined with similar kinetics until they were virtually undetectable. At all stages of development, trans -carveol dehydrogenase activity was 2 to 15 times greater than limonene synthase activity, and both were considerably higher than limonenehydroxylase activity.
The dotted line in Figure 4 B depicts the carvone accumulation rate, and represents the first derivative of the sigmoidal curve describing carvone accumulation in Figure 4 A. In general, there is a good correlation between the mathematically derived rate of carvone accumulation and the limonenehydroxylase activity measured in vitro, although the measured enzyme activity often underestimated the carvone accumulation rate in planta, especially during the first 15 DAP.
This enzyme also produces minor amounts 1. The properties of this enzyme resemble those of other monoterpenol dehydrogenases described in the literature. As a group, the monoterpenol dehydrogenases possess a wide range of pH optima, varying from around 8.
The trans -carveol dehydrogenase studied here also has a high pH optimum, around This finding is consistent with a trend noted earlier by Kjonaas et al. Nevertheless, the oxidation of monoterpenols in other plant species has been found to be catalyzed by a dehydrogenase activity other than the general alcohol ethanol dehydrogenase Croteau and Felton, This was confirmed for caraway by the absence of ethanol dehydrogenase activity at pH The three enzymes of monoterpene biosynthesis in caraway, limonene synthase, limonenehydroxylase, and trans -carveol dehydrogenase, undergo dramatic changes in activity during fruit development Fig.
During the early stages of fruit development 5—10 DAP there is an abundant accumulation of limonene, but very little carvone is produced. At this time, both the first and third enzymes of the pathway, limonene synthase and trans -carveol dehydrogenase, exhibit high levels of activity.
However, only trace levels of the second enzyme, limonenehydroxylase, were observed. The absence of significant amounts of limonenehydroxylase apparently prevents the oxidation of limonene to trans -carveol.
This blocks the formation of carvone, despite the consistently high levels of trans -carveol dehydrogenase activity, leading to a buildup of limonene. From 10 DAP, there is a rise in limonenehydroxylase activity that coincides with the appearance of substantial amounts of carvone.
Similar changes in terpenoid accumulation patterns occur during plant development in many other plant species, including dill Porter et al. However, to our knowledge, the enzymatic bases of such shifts in accumulation have been unexamined until now.
In contrast, the rapid switches in terpenoid metabolism that occur upon pathogen infection have been extensively investigated. For example, when cell cultures of tobacco or potato are treated with fungal elicitors, there is an induction of sesquiterpenoid phytoalexin biosynthesis and a repression of sterol formation Brindle et al.
Enzymatic analyses have revealed that fungal elicitors activate farnesyl diphosphate-utilizing enzymes in phytoalexin biosynthesis while reducing the activity of squalene synthase, a branchpoint enzyme in sterol formation that also competes for farnesyl diphosphate.
In caraway limonenehydroxylase may serve as an important rate-controlling step in carvone formation, not only because of its limited temporal occurrence, but also as a result of its kinetics. The hydroxylation of limonene to trans -carveol appears to be a much slower transformation than the subsequent dehydrogenation to carvone, as indicated by the lack of any accumulation of the intermediate, trans -carveol.
No 14 C-labeled trans -carveol was detected after [U- 14 C]Suc feeding in this study. In addition, analysis of the essential oil of caraway fruits has revealed only minute amounts of carveols: Monoterpene hydroxylation may also possess regulatory importance in the formation of monoterpenes in peppermint.
As peppermint leaves mature, the percentage of limonene drops and the percentage of the oxygenated products menthone and menthol increases Brun et al. The enzymatic changes responsible for this metabolic switch are currently under investigation M.
The correlation between limonenehydroxylase activity and the profile of carvone accumulation during fruit development is not exact Fig. For example, in vitro activity is frequently insufficient to account for carvone accumulation, probably because of difficulties in quantitatively extracting and assaying this enzyme.
Limonenehydroxylase is a Cyt Pdependent oxygenase Bolwell et al. The activities of enzymes of this class are often underestimated as a result of inefficient extraction and poor stability Mihaliak et al.
The formation of monoterpenes in caraway fruit may also be controlled by enzymes acting prior to limonene synthase, which control flux entering the monoterpene pathway Gershenzon and Croteau, At 15 to 20 DAP, the biosynthesis of limonene and carvone in developing fruits appears to cease, based on the lack of further [ 14 C]Suc incorporation Table III and the leveling off of the accumulation curves Fig.
Nevertheless, ample activities of all three enzymes of the pathway are still present, judging by the results of the in vitro assays Fig. At this stage of development, the formation of monoterpenes could be limited by substrate partitioning to competing pathways, such as triacylglycerol synthesis.
The rate of fatty acid formation in developing fruits increases dramatically at this stage Table III ; Fig. The biosynthesis of structural carbohydrates, which has been found to slightly precede triacylglycerol accumulation in caraway fruits Luyendijk, , may also compete for the supply of fixed carbon.
The accumulation patterns of monoterpenes and fatty acids during caraway fruit development and the profiles of enzyme activities involved in monoterpene biosynthesis were quite similar for the two caraway forms data not shown.
However, in annual caraway, accumulation of limonene and carvone started earlier in development, reached higher levels per fruit, and ceased earlier than in biennial caraway. These differences were partially reflected in the time courses of enzyme activity for limonene synthase and limonenehydroxylase.
Both activities were higher in annual than in biennial caraway at early stages of development. However, the biennial form continued to accumulate limonene and carvone after the monoterpene content of annual caraway had already stabilized.
The continued formation of monoterpenes in the later stages of development of biennial caraway may also explain the higher carvone-to-limonene ratio of this form. If competition for substrate is important in controlling the rate of monoterpene biosynthesis in caraway fruits, this phenomenon appears to be less important, or of later onset, in the biennial form.
The formation of monoterpenes in developing caraway fruits may be controlled by subcellular compartmentation of the various enzymes. The first enzyme of the pathway, limonene synthase, appears to be localized in the leukoplasts, colorless plastids with few internal membranes Gleizes et al.
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