Abstract: The present invention relates to a novel mutant of Aspergillus terreus which shows a resistance to both cerulenin and L-methionine analogue, and a process for preparing mevinolinic acid which comprises aerobic culture of the mutant strain and recovery of mevinolinic acid. The mutant of the present invention provides a remarkably high productivity of mevinolinic acid while reducing the production of byproducts such as mevinolinic acid analogues, when compared with a wild type Aspergillus terreus isolated from soil environment in Korea, and it successfully produce mevinolinic acid by employing monosaccharides such as glucose and galactose, unlike the mother strain.

Abstract: A process for the microbiological or enzymatic hydrolytic resolution of racemic trans-2-(alkoxycarbonylethyl) lactams of the formula I: ##STR1## wherein R is C.sub.1 -C.sub.7 alkyl, 2,2,2-trifluoroethyl or methoxyethoxyethyl and R.sup.1 is hydrogen or a protecting group is disclosed, whereby an optically enriched compound of the formula Ib or IIa: ##STR2## is obtained.

Abstract: The present invention is directed to an improved process for making PDE IV inhibitors. In specific, this application describes a process for making and purifying a chiral bisaryl alcohol, an intermediate compound necessary for the preparation of PDE IV inhibitors such as CDP 840, by asymmetric bioreduction of a pro-chiral ketone. Furthermore, the bioprocess provides for production of each enantiomer of a bisaryl alcohol at elevated optical purity.This invention takes advantage of a microorganism's ability to reduce a pro-chiral bisaryl ketone to the chiral bisaryl alcohol. The alcohol is readily isolated from the media by solvent extraction, crystallography, or other purification method known to the skilled artisan. The chiral alcohol can then be converted to a PDE IV inhibitor, such as CDP 840, by methods well known in the art.

Abstract: Method to produce a more active ribozyme by introducing a modified base into a substrate binding arm of the ribozyme or its catalytic core.

Abstract: The invention provides methods and compositions relating to a human telomerase and related nucleic acids, including four distinct human telomerase subunit proteins called p140, p105, p48 and p43 having human telomerase-specific activity. The proteins may be produced recombinantly from transformed host cells from the disclosed telomerase encoding nucleic acids or purified from human cells. Also included are human telomerase RNA components, as well as specific, functional derivatives thereof. The invention provides isolated telomerase hybridization probes and primers capable of specifically hybridizing with the disclosed telomerase gene, telomerase-specific binding agents such as specific antibodies, and methods of making and using the subject compositions in diagnosis, therapy and in the biopharmaceutical industry.

Abstract: Nucleic acids comprising the RNA component of a mouse, rat, Chinese hamster and bovine telomerase are disclosed, as are recombinant expression plasmids comprising said nucleic acids and host cells transformed with said recombinant expression plasmids.

Abstract: The present invention provides new methods, employing a nucleotide integrase, for cleaving double-stranded and single stranded DNA substrates at specific sites and for attaching nucleic acid molecules to the cleaved DNA substrates. One method uses a nucleotide integrase to cleave one strand of a double-stranded DNA and to concomitantly attach a nucleic acid molecule to the cleaved strand. Another method uses a nucleotide integrase to cleave both strands of a double-stranded DNA substrate and to attach a nucleic acid molecule to one strand of the DNA substrate. Another method uses a nucleotide integrase to cleave both strands of a double-stranded DNA substrate and to attach an RNA molecule to one strand of the substrate and for attaching a cDNA to the other strand of the substrate. Another method cleaves single stranded DNA with the concomitant insertion of a nucleic acid molecule at the cleavage point.

Abstract: A novel transposon useful for sequencing long DNAs is disclosed which comprises a partial sequence of transposon Tn5 with the oligonucleotide primers from phages SP6 and T7 inserted near the opposite ends, respectively, of said transposon Tn5.

Abstract: The present invention provides a method for detecting a mutation in a nucleic acid molecule which comprises contacting the nucleic acid molecule with a probe. The probe comprises two covalently linked nucleic acid segments under conditions such that the unlinked end of each segment of the probe is capable of hybridizing with the nucleic acid molecule. This mixture is then contacted with a ligase under conditions such that the two hybridized probe segments will ligate and bind the nucleic acid molecule if the nucleic acid molecule contains the mutation. One would then determine the presence of bound nucleic acid molecule(s) and thereby detect the mutation in the nucleic acid molecule.

Abstract: Purified and recombinant proteins TPC2 and TPC3 and recombinant or synthetic oligonucleotides corresponding to those proteins or fragments thereof can be used to detect regulators of telomere length and telomerase activity in mammalian cells and for a variety of related diagnostic and therapeutic purposes.

Abstract: Compositions and methods are provided for the treatment and diagnosis of diseases amenable to modulation of the production of selected proteins. In accordance with preferred embodiments, oligonucleotides and oligonucleotide analogs are provided which are specifically hybridizable with a selected sequence of RNA or DNA wherein at least one of the 2'-deoxyfuranosyl moieties of the nucleoside unit is modified. Treatment of diseases caused by various viruses and other causative agents is provided.

Abstract: A polypeptide consisting essentially of the NAB and linking region of an .alpha.-subunit of Shaker-like potassium ion channel which binds to a core region of a .beta.-subunit of said Shaker-like potassium ion channel. A related polypeptide is also provided and consists essentially of the core region of a .beta.-subunit of a Shaker-like potassium ion channel which binds to the NAB and linking region of an .alpha.-subunit of said Shaker-like channel. Nucleic acid sequences which encode these polypeptides, vectors containing those sequences, expression systems, hosts cells containing the aforesaid polypeptides, and pharmaceutical formulations of the peptides are also provided.Other aspects of the invention include methods of modulating the flow of potassium ions through a cell membrane surrounding a cytoplasm by introducing either of the aforesaid polypeptides or exogenous Kv.beta.

Abstract: Methods and expression constructs are provided for the cloning and overexpression of an arabinoxylan degrading enzyme of fungal origin in a selected microbial host cell. The enzyme is shown to be active in the degradation of water-insoluble solids obtained from maize. The enzyme can be used in the preparation of animal feed compositions, human food or in industrial processes.

Abstract: A process is disclosed for inhibiting the amplification of a DNA template by subjecting a sample of biological material containing nucleic acid to the polymerase chain reaction (PCR) using a DNA polymerase deficient in 5' exonuclease activity. The method comprises forming a PCR admixture comprising the DNA template, first and second oligonucleotide primers which are complementary to separated regions of the nucleic acid template, a non-extendable oligonucleotide blocker which is complementary to the inter-primer region of the DNA, and the DNA polymerase lacking 5' exonuclease activity, and subjecting the PCR admixture to at least one PCR thermocycle. The DNA polymerase lacking 5' exonuclease activity is incapable of excising the non-extendable blocker which anneals to the DNA template during the PCR, thereby inhibiting amplification which would otherwise occur during the PCR. Preferably, the DNA polymerase lacking 5' exonuclease activity is the Stoffel fragment of Taq polymerase.

Abstract: Methods of detecting the RNA component of telomerase, diagnosing cancer, and determining its prognosis using polynucleotides that hybridize to the RNA component of mammalian telomerase in a sample.

Abstract: The present invention and kits are directed to a method of amplifying and detecting single or double-stranded target nucleic acid molecules in a test sample. Amplification is accomplished through the use of a minimum of two oligonucleotide probe complement pairs, wherein members oligonucleotide probes from both pair of oligonucleotide probe complement pairs form a minimum of two oligonucleotide probe pairs, at least one of which is complementary to a given portion of a target nucleic acid sequence which act as template. One of the oligonucleotide probes of each oligonucleotide probe pair have an additional protecting sequence which is not complementary to the target sequence. These additional protecting sequences are preferably complementary to each other. Chemical functionality groups attached to the oligonucleotide probes covalently combine the probes to form a joined oligonucleotide product. The joined oligonucleotide product is formed without the use of enzymes.

Abstract: The present invention features a method for introducing hypermutations into a target DNA or RNA sequence of interest, characterized in that said method comprises the steps of:(a) transcribing a RNA into DNA in a reaction mixture comprising a reverse transcriptase, varying biased concentrations of deoxynucleoside triphosphates to produce hypermutations and an oligonucleotide primer that is partially complementary to the 3' end of said RNA; and(b) recovering said DNA sequences.

Abstract: The invention provides devices and methods for use in detecting nucleic acid analytes in samples. The devices each include a solid support to which is bound a two-dimensional distribution or field of nucleic acid probes that each bind to a nucleic acid analyte, which is detected by use of amplification methods.

Abstract: The present invention is directed toward efficient, high-yield processes for making ascorbic acid, 2-keto-L-gulonic acid, and esters of 2-keto-L-gulonic acid. The processes comprise reacting the appropriate starting materials with a hydrolase enzyme catalyst such as a protease, an esterase, a lipase or an amidase.

Abstract: Provided herein is a rapid method for isolating total RNA from test samples and further for isolating mRNA from test samples.

Abstract: A FO-1289 substance of the formula ##STR1## wherein R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are hydrogen or acyl. A process for the production thereof comprises culturing the novel microorganism Aspergillus sp. FO-1289 FERM BP-4242 in a nutrient medium, accumulating the FO-1289 substance thus produced in the medium, and isolating the FO-1289 substance therefrom. Particular species of the FO-1289 substance comprise FO-1289A, in which R.sub.1, R.sub.2 and R.sub.3 are acetyl and R.sub.4 is hydrogen; FO-1289B, wherein R.sub.1 is propionyl, R.sub.2 and R.sub.3 are acetyl and R.sub.4 is hydrogen; FO-1289C, wherein R.sub.1 and R.sub.3 are acetyl, R.sub.2 is propionyl and R.sub.4 is hydrogen; and FO-1289D, wherein R.sub.1 and R.sub.2 are acetyl, R.sub.3 is propionyl and R.sub.4 is hydrogen.

Abstract: An improved method for the simultaneous sequence-specific identification of mRNAs in a mRNA population allows the visualization of nearly every mRNA expressed by a tissue as a distinct band on a gel whose intensity corresponds roughly to the concentration of the mRNA. In general, the method comprises the formation of cDNA using anchor primers to fix a 3'-endpoint, producing cloned inserts from the cDNA in a vector containing a bacteriophage-specific promoter for subsequent RNA synthesis, generating linearized fragments of the cloned inserts, preparing cRNA, transcribing cDNA from the cRNA using a set of primers, and performing PCR using a 3'-primer whose sequence is derived from the vector and a set of 5'-primers that is derived from the primers used for transcription of cDNA from cRNA. The method can identify changes in expression of mRNA associated with the administration of drugs or with physiological or pathological conditions.

Abstract: Internal Transcribed Spacer (ITS) DNA sequences from the ribosomal RNA gene region are described for different species and strains of Helminthosporium carbonum, Helminthosporium turcicum, Helminthosporium maydis, Cercospora zeae-maydis, Kabatiella zeae and Puccinia sorghi. Specific primers from within these sequences are identified as being useful for the identification of the fungal isolates using PCR-based techniques.

Abstract: The invention relates to a system for transporting nucleic acids into the cell, which is effected by receptor-mediated endocytosis. Using a transferrin-polycation conjugate, a complex can be formed with the polyanionic nucleic acid. This complex is bound to the transferrin receptor, which is highly regulated in growing cells, and absorbed into the cell. Suitable nucleic acids include those which inhibit specific genes or the RNA function, such as antisense oligonucleotides or ribozymes or the genes coding for them. The invention further relates to a process for introducing nucleic acids into the cells, transferrin-polycation/nucleic acid complexes and pharmaceutical preparations containing them.

Abstract: Methods are provided for substantially reducing background signals encountered in nucleic acid hybridization assays. The method is premised on the elimination or significant reduction of the phenomenon of nonspecific hybridization, so as to provide a detectable signal which is produced only in the presence the target polynucleotide of interest. In addition, a novel method for the chemical synthesis of isoguanosine or 2'-deoxy-isoguanosine is provided. The invention also has applications in antisense and aptamer therapeutics and drug discovery.

Abstract: The present invention makes available methods and reagents for novel manipulation of nucleic acids. As described herein, the present invention makes use of the ability of intronic sequences, such as derived from group I, group II, or nuclear pre-mRNA introns, to mediate specific cleavage and ligation of discontinuous nucleic acid molecules. For example, novel genes and gene products can be generated by admixing nucleic acid constructs which comprise exon nucleic acid sequences flanked by intron sequences that can direct trans-splicing of the exon sequences to each other. The flanking intronic sequences can, by intermolecular complementation, form a reactive complex which promotes the transesterification reactions necessary to cause the ligation of discontinuous nucleic acid sequences to one another, and thereby generate a recombinant gene comprising the ligated exons.

Abstract: A prototype RNA cyclase ribozyme that allows efficient production of circular RNA. Methods for modifying the prototype to produce a wide variety of custom circular RNA are detailed. The method utilizes a new plasmid which enables production of a wide variety of imaginable RNA sequences in a covalent, circular form free from intron sequences in vitro. At a particular site in the plasmid, a sequence coding for the desired circular RNA is inserted to create a new RNA cyclase ribozyme gene. RNA transcribed from RNA cyclase ribozyme genes autocatalytically converts the desired RNA sequence it contains into circular form. RNA cyclase genes may be placed into appropriate expression vectors for synthesis of circular RNA in vivo as part of ribozyme or antisense gene regulation approaches to genetic engineering.

Abstract: The initial steps in photosynthesis are the conversion of light energy into chemical energy. This conversion is performed by the multisubunit protein-pigment complexes of the thylakoid membranes. Oxygen-evolving photosystems contain photosystem I (PSI) and photosystem II (PSII), which act in tandem. In PSII, the majority of light-adsorbing chlorophylls are attached to LHCII, the light harvesting complex associated with this photosystem. LHCII is the most abundant member of the family of chlorophyll a/b binding (CAB) proteins. A gene encoding a type I chlorophyll a/b binding protein of LHCII (ICABPSII) has been cloned from Brassica napus L. An anti-sense RNA of this gene has been used to reduce the amount of chlorophyll a/b binding protein and thus reduce the amount of chlorophyll in the resulting transgenic plants. By using "site" specific promoters the reduction of chlorophyll can be targeted to specific organelles in the transgenic plant and thus can be used to reduce the green color at these sites.

Abstract: The present invention relates to a product and process for producing polypeptides, such products including a circular RNA having a ribosome binding site that engages an eukaryotic ribosome and cells transformed with such circular RNA. Circular RNA is produced by linking the 5' and 3' ends of a desired linear RNA sequence, and such constructs can be used to produce desired amounts of polypeptides when such constructs are translated either in vitro or in vivo. The present invention also relates to the use of circular RNA as a pharmaceutical agent to treat cells and animals involved in a disease.

Abstract: Methods and compositions are described for making ribozymes which can release or activate molecules including autocatalytically replicatable RNA such as MDV-1.

Abstract: The present invention provides recombinant polynucleotides comprised of elements that regulate transcription and/or expression of coding sequences. These regulatory elements have been isolated from a CD69 gene, and thus are of particular use in regulating transcription and/or expression in cells which express CD69.

Abstract: The present invention relates to methods of synthesizing phosphorothioate oligonucleotides. In particular, it relates to the use of certain restriction endonucleases to cleave phosphorothioate oligonucleotides which contain restriction endonuclease recognition sequences. These restriction sequences facilitate the cleavage of relatively cleavage resistant phosphorothioate oligonucleotides thus facilitating their separation and purification after synthesis.

Abstract: The present invention is directed to a family of phospholipid binding and transporting proteins, a method for preparing same from fungi, and their use in cosmeticology, the agri-foodstuffs industry and pharmacology Phospholipid proteins are capable of binding, transporting and/or rearranging lipids between membranes, optionally in combination with active principles. Furthermore, the phospholipid proteins are hydrophobic and acidic, have a molecular weight of under 50 kDa, and may be prepared from a non-toxic filamentous fungus capable of developing on a lipid-enriched medium, particularly from raw extracts of fungi.

Abstract: The oligoribonucleotide analogs of the invention are relatively small, three-dimensional structures derived from larger parental RNA molecules. The analogs include a first nucleic acid structure including one or more nucleotide sequences that are derived from a region of parental RNA, wherein in its native state, the region binds to a ligand, e.g., an aminoglycoside, with a parental RNA ligand binding pattern, and a second nucleic acid structure including one or more nucleotide sequences combined with the first nucleic acid structure to form the analog and provide the analog with a conformation that binds the ligand with a ligand binding pattern that is substantially identical to the parental RNA ligand binding pattern. These analogs can be used to identify novel therapeutic compounds.

Abstract: A method is provided for preferentially amplifying target RNA relative to non-target RNA in a sample of tester RNA. According to the method, a sample of tester RNA is contacted with driver sequences which are complementary to the non-target RNA under conditions where the driver sequences hybridize to the non-target RNA. A nucleic acid primer is then extended using the target RNA as a template, forming a DNA template complementary to some or all of the target RNA. The DNA template formed using the target RNA as template is then rendered single-stranded to enable the hybridization of the DNA template to a promoter template. The DNA template is then extended using the promoter template to form an extended DNA template comprising a functional double-stranded promoter. Multiple copies of the target RNA sequence can now be transcribed from the extended DNA template through recognition of the contained functional double-stranded promoter by RNA polymerase.

Abstract: Methods and kits for relating initial amounts of target nucleic acids present in a sample to target-specific amplification products. It has been discovered that the transcription-mediated amplification system is capable of producing a quantitative relationship between target input and target-specific output. Further, the present invention relates to methods for carefully controlling this relationship resulting in an unexpectedly high degree of reproducability. Also described are useful methods for extending the dynamic range of transcription-based amplification systems.

Abstract: Nucleic acid probes and primers are described for detecting fungi that cause disease in humans and animals, as well as spoilage of food and beverages. These probes can detect rRNA, rDNA or polymerase chain reaction products from a majority of fungi in clinical, environmental or food samples. Nucleic acid hybridization assay probes specific for Acremonium sp., Aspergillus clavatus, Aspergillus flavus, Aspergillus fumigatus, Aspergillus glaucus, Aspergillus nidulans, Aspergillus niger, Aspergillus ochraceus, Aspergillus terreus, Aspergillus unguis, Aspergillus ustus, Beauveria sp., Bipolaris sp., Blastoschizomyces sp., Blastomyces dermatitidis, Candida albicans, Candida glabrata, Candida guilliermondii, Candida kefyr, Candida krusei, Candida lusitaniae, Candida parapsilosis, Candida tropicalis, Chrysosporium sp., Cladosporium sp., Coccidioides immitis, Cryptococcus neoformans var gattii serotype B, Cryptococcus neoformans serotype A, Cryptococcus laurentii, Cryptococcus terreus, Curvularia sp., Fusarium sp.

Abstract: The instant invention provides a method for producing RNA enzymatically in a semi-batch or continuous-flow bioreactor using a DNA template immobilized to a solid support through a linkage having a single-stranded overhang extending from a noncoding strand. The coding strand remains dissociable from the immobilized noncoding strand. The immobilized RNA template is reused in at least sixteen rounds of transcription reaction. The coding strand can be replaced with a fresh DNA strand encoding the same or a different RNA.

Abstract: There is provided a process for production of carane-3,4-diol which includes contacting a culture of filamentous fungus having an ability to produce carane-3,4-diol from 3-carene or 3,4-epoxycarane, cells collected from the culture or treated cells with 3-carene or 3,4-epoxycarane, then recovering the resulting carane-3,4-diol.

Abstract: A metabolite, e.g., ethanol, is continuously produced from low cost carbohydrate substrates by a process which comprises pulverizing the carbohydrate substrate; liquefying and saccharifying the pulverized substrate; continuously fermenting the lique-saccharified substrate in a fermentor equipped with a moving filter, in the presence of flocculent biological cells maintained at a concentration ranging from 90 to 160 g/l by using the moving filter and a culture medium to produce a fermentation product mixture; and recovering the desired metabolite from the fermentation product mixture.

Abstract: Methods are disclosed for producing a sequence tag from a polynucleotide. In preferred embodiments, the method comprises (a) digesting the polynucleotide with a type II restriction enzyme to produce a first cohesive end; (b) providing a promoter-linker cassette comprising an RNA polymerase promoter, a restriction site for a type IIS restriction enzyme and a second cohesive end which is complementary to the first cohesive end produced by the type II restriction enzyme; (c) ligating the digested polynucleotide to the cassette by joining the first cohesive end and the second cohesive end; (d) digesting the ligated product of step (c) with the type Iis restriction enzyme; and (e) transcribing the resulting construct from the promoter thereby producing a sequence tagged polyribonucleotide.

Abstract: There are provided nucleic acid hybridization assays for RNA targets using RNA binary probes and a ribozyme ligase that is a stringent RNA-directed RNA ligase. Preferred assays include exponential amplification for signal generation. Tetrahymena ribozyme ligase is a preferred ligase for use in this invention. It may be tethered to hold it close to the ligation junction. One assay according to this invention is a "tethered ligase chain reaction." Also provided are kits for performing assays according to the invention.

Abstract: A reagent, method and container for the isolation of cellular components such as ribonucleic acid (RNA) from cells in a liquid solution. The container includes a cover assembly and a holder which is normally closed by the cover assembly and contains an RNA extractant solvent, micron-sized particles and at least one larger particle suitably of millimeter size. The container contains the reagent, which is an extractant solvent which contains phenol and guanidinium thiocyanate or guanidinium chloride and has a pH of about 4. The container also includes a friable sealing layer which separates the extractant solvent from the liquid medium containing the cells until the container is reciprocally shaken. The method includes the reciprocal shaking of the container, wherein the larger particle breaks the friable layer to permit mixing of the liquid medium with the extractant solvent resulting in the breaking of the cell walls by the micron-size beads and the release of the RNA.

Abstract: A method for preparing a ds DNA from a ss DNA template, which method includes:(a) providing a first DNA strand;(b) adding a homopolymeric oligonucleotide tail to the 3' end of the first DNA strand, to yield a tailed first DNA strand;(c) providing a ss homopolymeric oligonucleotide primer complementary to a portion of the tail;(d) contacting the primer with the tailed first DNA strand;(e) synthesizing, in the presence of the primer and the tailed first DNA strand, a second DNA strand complementary to the first DNA strand; and(f) removing the tail and the primer from the first and second DNA strands, respectively; provided that one or both of the tail and the primer contain(s) RNA.

Abstract: Itaconic acid and/or salt thereof is produced via aerobic microbial fermentation, for example by means of the species Aspergillus terreus or Aspergillus itaconicus, of a nutrient medium containing a source of assimilable carbon, such carbon source at least in part comprising an effective amount of glycerol.

Abstract: A method of site-directed alteration (removal or removal followed by replacement) of selected nucleotides in an RNA molecule, as well as to mixed phosphate backbone oligonucleotides useful in the method. It further relates to a method of producing polypeptides or proteins encoded by the RNA molecule altered by the present method. Through use of the present method, site-directed cleavage of an RNA molecule is effected, followed by excision of the selected or target segment of the RNA molecule.

Abstract: A novel multi-unit ribozyme is provided which is capable of selectively cleaving the mRNA transcript of a hybrid oncogene resulting from a chromosomal translocation. Specialized delivery vehicles such as liposomes, and growth factor conjugates for cellular uptake by receptor-mediated endocytosis, are also described. The multi-unit ribozyme is used for the treatment of neoplasms characterized by expression of the oncogene. In one embodiment, a multi-unit ribozyme is provided which cleaves the mRNA transcript of Philadelphia chromosome-positive cells, thereby blocking production of the tumorigenic p210.sup.bcr-abl fusion protein.

Abstract: This invention relates to the use of functional reporter molecules in the detection and measurement of RNA sequences in a sample, as a determination, for example, of pathogenic disease existence or potential. The invention is predicated on the utilization of nucleotide sequences, one having a probe sequence linked to a sequence capable of initiating replication by an RNA-dependent RNA polymerase. The other is capable of hybridizing to a strand separated from the extension product of the first nucleotide sequence after hybridization to a specific target sequence. The extension product of the second hybridized nucleotide sequence serves as a template source for autocatalytic replication by the RNA-dependent RNA polymerase. The replication products are detected as a means for detection of nucleic acid sequences.

Abstract: This invention concerns recombinant RNA molecules comprising a recognition sequence for the binding of an RNA-directed RNA polymerase, a sequence for the initiation of product strand synthesis and a heterologous sequence of interest inserted at a specific site in the internal region of the recombinant molecule. Such recombinant RNA molecules are capable of serving as a template for the synthesis of complementary single-stranded molecules by RNA-directed RNA polymerase. The product molecules so formed are also capable of serving as a template for the synthesis of additional copies of the original recombinant RNA molecule. In a preferred embodiment of the invention Q.beta. replicase is used as the RNA-directed RNA polymerase.

Abstract: A method and kit for purification of m-RNA from a cell are disclosed. Guanidine containing moieties, at high molarity, are use to quickly lyse the cell. They also act to inhibit RNase activity. Without the need for isolation of total RNA, the lysate can then be directly purified from the lysate using oligo dT (or U) by reducing the guanidine concentration via a dilution step.