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At least 1,387 records · Page 77Linked to original sources
PubMed2003

A matrix metalloproteinase 2 cleavable melittin/avidin conjugate specifically targets tumor cells in vitro and in vivo.

Extracellular matrix breakdown as well as increased expression in cancer cells and tumor microvascular endothelial cells make matrix metalloproteinase 2 (MMP2) an attractive target for cancer treatment. By taking advantage of MMP2's properties, an MMP2 cleavable melittin/avidin conjugate was designed. Melittin alone is extremely toxic to cells and induces immediate cell lysis, but becomes inactive when coupled with avidin. The incorporation of the MMP2 target sequence into the peptide was used as a means for targeting tumor cells. In vitro, the melittin/avidin conjugate showed strong cytolytic activity against cancer cells with high MMP2 activity; DU 145 prostate cancer cells and SK-OV-3 ovarian cancer cells. The conjugate exhibited very little cytolytic activity against normal L-cells that displayed low MMP2 activity. These data demonstrate the MMP2 specificity of the melittin/avidin conjugate. In vivo, the size of tumors injected with the melittin/avidin conjugate was significantly smaller as compared to untreated tumors. Therefore, due to its tumor targeting capabilities as well as its cytolytic properties in vitro and in vivo, the melittin/avidin conjugate displays the potential for use in cancer therapy.

Amino Acid Sequence↗
PubMed1990

TAR-independent activation of the HIV-1 LTR: evidence that tat requires specific regions of the promoter.

Replication of HIV-1 requires Tat, which stimulates gene expression through a target sequence, TAR. It is known that TAR is a Tat-responsive target. Since Tat increases transcriptional initiations from the HIV-1 LTR promoter, it is unclear mechanistically how Tat utilizes an RNA target. Here we show that TAR RNA is only one component of the Tat-responsive target. Efficient Tat trans-activation was observed only when TAR was present in conjunction with the HIV-1 LTR NF-kappa B/SP1 DNA sequences. TAR RNA outside of this context produced a suboptimal Tat response. We propose that TAR RNA serves an attachment function directing Tat to the LTR. A Tat protein engineered to interact with LTR DNA could trans-activate through a TAR-independent mechanism. This suggests that Tat also has a DNA target.

Animals↗
PubMed1998

Activity of foreign proteins targeted within the bacteriophage T4 head and prohead: implications for packaged DNA structure.

The phage-derived expression, packaging, and processing (PEPP) system was used to target foreign proteins into the bacteriophage capsid to probe the intracapsid environment and the structure of packaged DNA. Small proteins with minimal requirements for activity were selected, staphylococcal nuclease (SN) and green fluorescent protein (GFP). These proteins were targeted into the T4 head by means of IPIII (internal protein III) fusions or CTS (capsid targeting sequence) fusions. Additional evidence is provided that foreign proteins are targeted into T4 by the N-terminal ten amino acid residue consensus CTS of IPIII identified in previous work. Fusion proteins were produced within host bacteria by expression from plasmids or by produc tion from recombinant phage carrying the fusion genes. Packaged fusion proteins CTS IPIII SN, CTS IPIII TSN, CTS IPIII GFP, CTS IPIII TGFP, and CTS GFP, where [symbol: see text] indicates a linkage peptide sequence Leu(Ile)-N-Glu cleaved by the T4 head morphogenetic proteinase gp21 during head maturation, are observed to exhibit intracapsid activity. SN activity within the head is demonstrated by loss of phage viability and by digested genomic DNA patterns visualized by gel electrophoresis when viable phage are incubated in Ca2+. Green fluorescent phage result immediately after packaging GFP produced at 30 degreesC and below, and continue to give green fluorescence under 470 nm light after CsCl purification. Non-fluorescent GFP-fusions are produced in bacteria at 37 degreesC, and phage packaged with these proteins achieve a fluorescent state after incubation for several months at 4 degreesC. GFP-packaged phage and proheads analyzed by fluorescence spectroscopy show that the mature head and the DNA-empty prohead package identical numbers of GFP-fusion proteins. Encapsidated GFP and SN can be injected into bacteria and rapidly exhibit intracellular activity. In vivo SN digestion of encapsidated DNA gives an intriguing pattern of DNA fragments by gel analysis, predominantly a repeat pattern of 160 bp multiples, reminiscent of a nucleosome digestion ladder, This quasi-limit DNA digestion pattern, reached >100-fold more slowly than the loss of titer, is invariant over a range </=10 to 200 molecules of SN packaged per head, and independent of proteolytic cleavage of SN from the IPIII portion of the fusion, favoring a discontinuous packaged DNA structure. Rods of B-form DNA could be envisioned as protected from digestion, whereas bent or kinked DNA would be more susceptible to the diffusible SN. Such discontinuous packaged DNA structures are favored for phage T4 by a number of lines of evidence.

Bacteriophage T4↗
PubMed1993

Ligation amplification and fluorescence detection of Mycobacterium tuberculosis DNA.

Current methods for the identification of Mycobacterium tuberculosis are dependent upon culture of the bacteria and are necessarily lengthy due to the slow growth of this agent. The development of DNA probe technology offers rapid, accurate and cost effective alternatives for the identification of such fastidious organisms. A technique for detecting specific DNA sequences, known as oligonucleotide ligation assay (OLA) involves the ligation of two adjacent oligonucleotides annealed to target DNA, and has been previously described. Amplification of the target sequences can be accomplished by including complementary pairs of oligonucleotides and a thermal stable ligase in a reaction which cycles between annealing/ligation and denaturing temperatures. Using a cloned portion of an insertion sequence, IS6110, which has been reported to be specific for M. tuberculosis complex as target DNA, we demonstrate the ligation dependent amplification of a 40 base pair region of plasmid bearing IS6110. By employing oligonucleotides which are each labelled with a different fluorescent dye, the reaction can be followed by fluorescence detection on an Applied Biosystems model 373A DNA sequencer. Using this approach, we have optimized conditions for the detection of 100 target molecules in a mixture containing 4 micrograms of unrelated DNA. Since the insertion sequence is repeated on average 12-14 times in the genome of M. tuberculosis, this corresponds to a theoretical detection level of 7-8 organisms. Completion of this entire assay can be accomplished in less than 8 h and serves as a basis for further studies in the development of a rapid clinical diagnostic test for tuberculosis.

Base Sequence↗
PubMed2006

Polyamide platinum anticancer complexes designed to target specific DNA sequences.

Two new platinum complexes, trans-chlorodiammine[N-(2-aminoethyl)-4-[4-(N-methylimidazole-2-carboxamido)-N-methylpyrrole-2-carboxamido]-N-methylpyrrole-2-carboxamide]platinum(II) chloride (DJ1953-2) and trans-chlorodiammine[N-(6-aminohexyl)-4-[4-(N-methylimidazole-2-carboxamido)-N-methylpyrrole-2-carboxamido]-N-methylpyrrole-2-carboxamide]platinum(II) chloride (DJ1953-6) have been synthesized as proof-of-concept molecules in the design of agents that can specifically target genes in DNA. Coordinate covalent binding to DNA was demonstrated with electrospray ionization mass spectrometry. Using circular dichroism, these complexes were found to show greater DNA binding affinity to the target sequence: d(CATTGTCAGAC)(2), than toward either d(GTCTGTCAATG)(2,) which contains different flanking sequences, or d(CATTGAGAGAC)(2), which contains a double base pair mismatch sequence. DJ1953-2 unwinds the DNA helix by around 13 degrees , but neither metal complex significantly affects the DNA melting temperature. Unlike simple DNA minor groove binders, DJ1953-2 is able to inhibit, in vitro, RNA synthesis. The cytotoxicity of both metal complexes in the L1210 murine leukaemia cell line was also determined, with DJ1953-6 (34 microM) more active than DJ1953-2 (>50 microM). These results demonstrate the potential of polyamide platinum complexes and provide the structural basis for designer agents that are able to recognize biologically relevant sequences and prevent DNA transcription and replication.

Animals↗
PubMed2001

Molecular cloning and characterization of WdPKS1, a gene involved in dihydroxynaphthalene melanin biosynthesis and virulence in Wangiella (Exophiala) dermatitidis.

1,8-Dihydroxynaphthalene (1,8-DHN) is a fungal polyketide that contributes to virulence when polymerized to 1,8-DHN melanin in the cell walls of Wangiella dermatitidis, an agent of phaeohyphomycosis in humans. To begin a genetic analysis of the initial synthetic steps leading to 1,8-DHN melanin biosynthesis, a 772-bp PCR product was amplified from genomic DNA using primers based on conserved regions of fungal polyketide synthases (Pks) known to produce the first cyclized 1,8-DHN-melanin pathway intermediate, 1,3,6,8-tetrahydroxynaphthalene. The cloned PCR product was then used as a targeting sequence to disrupt the putative polyketide synthase gene, WdPKS1, in W. dermatitidis. The resulting wdpks1Delta disruptants showed no morphological defects other than an albino phenotype and grew at the same rate as their black wild-type parent. Using a marker rescue approach, the intact WdPKS1 gene was then successfully recovered from two plasmids. The WdPKS1 gene was also isolated independently by complementation of the mel3 mutation in an albino mutant of W. dermatitidis using a cosmid library. Sequence analysis substantiated that WdPKS1 encoded a putative polyketide synthase (WdPks1p) in a single open reading frame consisting of three exons separated by two short introns. This conclusion was supported by the identification of highly conserved Pks domains for a beta-ketoacyl synthase, an acetyl-malonyl transferase, two acyl carrier proteins, and a thioesterase in the deduced amino acid sequence. Studies using a neutrophil killing assay and a mouse acute-infection model confirmed that all wdpks1Delta strains were less resistant to killing and less virulent, respectively, than their wild-type parent. Reconstitution of 1,8-DHN melanin biosynthesis in a wdpks1Delta strain reestablished its resistance to killing by neutrophils and its ability to cause fatal mouse infections.

Amino Acid Sequence↗
PubMed2001

CD and melting curves structural studies of the tandem DNA complex formed with oligonucleotides carrying photoactive and sensitizing groups in the nick region.

Photoactive derivatives of oligonucleotides are widely used as affinity reagents for the study of structures and functions of nucleic acids and proteins. Between them the binary reagents are the more attractive in the last time. They represent the tandem of two oligonucleotide derivatives complementary to a target sequence and carrying photoactive and sensitizing groups. The efficiency of target modification in this case depends on the mutual arrangement in the nick region of photoactive and sensitizing groups, attached to the oligonucleotides. The use of binary reagents in affinity modification permits to reach the high selectivity of the process. In this work we report our studies on the thermodynamic and structural peculiarities of complementary tandem complex between DNA target and binary oligonucleotide reagent. The complex consisted of the target d(TTGAAGGGGACCGC)and two 7-mer oligonucleotide conjugates,one of which was modified on its 3'-phosphate with a photoreactive p-azidote-trafluorobenzaldehydehydrazone-group,and the other one was linked through its 5'-phosphate to a sensitizing perylene-group. Optical melting curves and thermal changes in circular dichroism (CD)spectra were detected for all possible oligonucleotide and/or conjugate combinations.In addition,molecular modeling simulation of the complex structure was carried out. It was found that CD spectra did not show serious changes in the B-helix structure of the duplex. The interaction between perylene-and azido-groups at the oligonucleotide junction led to considerable increase in duplex stability. CD and molecular modeling data clearly indicated that perylene-group interacted with the duplex in an intercalative manner,but azido-group located on the side of DNA chain minor groove.

Azides↗
PubMedDate not supplied

Time-resolved fluorometry in end-point and real-time PCR quantification of nucleic acids.

Two time-resolved fluorescence-based methods for nucleic acid quantification are described and their results are compared. Both methods use an exogenous internal standard to eliminate errors arising from different steps of the assay. The first method is a competitive end-point assay, where the standard competes for the same primers with the actual target sequence, prostate-specific antigen (PSA) cDNA. The standard and target are quantified in a dual-label plate hybridization with lanthanide-labelled probes after a fixed number of PCR cycles. The second method is based on real-time monitoring of PCR and on the use of a novel homogeneous signal generation principle that relies on the use of a 5'-->3' exonucleolytic DNA polymerase and a probe labelled with an environment sensitive, stable and fluorescent lanthanide chelate. In this assay, a non-competitive, exogenous internal standard is used. Both assays have a wide linear range (50-5 x 10(6) and 10-5 x 10(7) input PSA cDNA molecules for the end-point and real-time assays, respectively) and there is a strong correlation between the results obtained with the two assays (r = 1.0). Being somewhat faster to perform, the real-time format is better suited for assays that require high throughput.

Base Sequence↗
PubMed1996

Mutations in target DNA elements of yeast HAP1 modulate its transcriptional activity without affecting DNA binding.

The yeast zinc cluster protein HAP1, a member of the GAL4 family, is a transcriptional activator that binds as a homodimer to target DNA sequences. These targets include the upstream activating sequences of the CYC1 and CYC7 genes, which have no obvious sequence similarity. Even though both sites have the same affinity for HAP1, activation differs at these two sites, even when the sequences are placed in an identical promoter context. In addition, mutants of HAP1 that can bind to both sites but are specifically transcriptionally inactive at CYC7 have been previously isolated. In order to identify nucleotides that are responsible for this differential activity, we have performed random and site-directed mutagenesis of these target sites and assayed their binding to HAP1 in vitro and their activity in vivo in reporter plasmids. Our results show that HAP1 binding sites are degenerate forms of the direct repeat CGG N3 TA N CGG N3 TA. Moreover, we show that activity of HAP1 mutants defective for activation of the CYC7gene is restored by specific mutations in the CYC7 binding site. Conversely, other mutations of the target sites prevent activation by HAP1, without interfering with DNA binding. The results suggest that the sequence of the target sites influences the conformation and, hence, the activity of DNA-bound HAP1.

Base Sequence↗
PubMed1996

Kinetic selectivity of complementary nucleic acids: bcr-abl-directed antisense RNA and ribozymes.

Efficacy and sequence specificity are two major requirements in the use of antisense nucleic acids and ribozymes. For long-chain complementary RNA sequences (>30 nt), effects in living cells are correlated with the association rate of the complementary RNA in vitro, but not with the stability of the formed double strand. Thus, sequence selectivity of complementary RNA has to be defined as fast versus slow annealing with the appropriate target or non-target sequences, respectively. In this work, we performed a systematic kinetic analysis to evaluate the selectivity of bcr-abl-directed antisense RNA and hammerhead ribozymes with a length of the complementary sequences of between 20 and 80 bases. By kinetic in vitro selection, we identified oligomeric as well as long-chain complementary RNA that annealed at least tenfold faster with the bcr-abl sequence in comparison with either of the wild-type sequences bcr or abl, respectively. In the presence of selected oligodeoxynucleotide sequences and RNase H, the bcr-abl transcript was specifically hydrolysed out of a mixture containing abl and bcr sequences as well. Hammerhead ribozymes were designed such that binding with their target was facilitated either via helix I or helix III-forming antisense arms but not both. Further, cleavage and binding occurred on opposite sides of the bcr-abl fusion point. Target selectivity was found for a ribozyme that annealed fast via abl sequences and cleaved within the bcr portion of bcr-abl RNA. Kinetic probing and calculations of the local folding potential indicate that the bcr-abl fusion point sequences are not easily accessible for complementary nucleic acids. This study supports the need for more detailed structural investigations of the bcr-abl fusion sequence and forms a more rational basis for the therapeutic use of nucleic acid inhibitors of the aberrant bcr-abl gene expression in Philadelphia chromosome-positive cells.

Base Sequence↗
PubMed2000

Octamer-primed sequencing technology: development of primer identification software.

Octamer sequencing technology (OST) is a primer-directed sequencing strategy in which an individual octamer primer is selected from a pre-synthesized octamer primer library and used to sequence a DNA fragment. However, selecting candidate primers from such a library is time consuming and can be a bottleneck in the sequencing process. To accelerate the sequencing process and to obtain high quality sequencing data, a computer program, electronic OST or eOST, was developed to automatically identify candidate primers from an octamer primer library. eOST integrates the base calling software PHRED to provide a quality assessment for target sequences and identifies potential primer binding sites located within a high quality target region. To increase the sequencing success rate, eOST includes a simple dynamic folding algorithm to automatically calculate the free energy and predict the secondary structure within the template in the vicinity of the octamer-binding site. Several parameters were found to be important, including base quality threshold, the window size of the template sequence segment, and the threshold [Delta] G value. OST, coupled with the eOST software, can be used to sequence short DNA fragments or in the finishing assembly stage of large-scale sequencing of genomic DNA.

Algorithms↗
PubMed1987

Organization of multispecific DNA methyltransferases encoded by temperate Bacillus subtilis phages.

B. subtilis phage rho 11s codes for a multispecific DNA methyltransferase (Mtase) which methylates cytosine within the sequences GGCC and GAGCTC. The Mtase gene of rho 11s was isolated and sequenced. It has 1509 bp, corresponding to 503 amino acids (aa). The enzyme's Mr of 57.2 kd predicted from the nucleotide sequence was verified by direct Mr determinations of the Mtase. A comparison of the aa sequence of the rho 11s Mtase with those of related phages SPR and phi 3%, which differ in their methylation potential, revealed generalities in the building plan of such enzymes. At least 70% of the aa of each enzyme are contained in two regions of 243 and 109 aa at the N and C termini respectively, which are highly conserved among the three enzymes. In each enzyme, variable sequences separate the conserved regions. Variability is generated through the single or multiple use of related and unrelated sequence motifs. We propose that the recognition of those DNA target sequences, which are unique for each of the three enzymes, is determined by these variable regions. Evolutionary relationships between the three enzymes are discussed.

Amino Acid Sequence↗
PubMed1989

Epstein-Barr virus BZLF1 trans-activator specifically binds to a consensus AP-1 site and is related to c-fos.

Two regions of the Epstein-Barr virus BZLF1 trans-activator protein have sequence similarity to the c-fos protein. Part of the similarity corresponds to the region of c-fos which is similar to the DNA binding domain of c-jun and GCN-4. The structure of the exon which contains this region in c-fos and BZLF1 is also highly conserved between the two genes. Complete BZLF1 protein and a C terminal fragment were prepared either as purified fusion proteins or by in vitro translation from a BZLF1 cDNA. Gel retardation and DNase footprinting assays using these proteins show that BZLF1 is a sequence specific DNA binding protein capable of binding to a target sequence which contains a consensus AP-1 site.

Amino Acid Sequence↗
PubMed2001

Sex determination in cattle based on simultaneous amplification of a new male-specific DNA sequence and an autosomal locus using the same primers.

A PCR-based method for sex determination of bovine DNA samples and embryo biopsies is presented. Using only one primer pair both the male-specific sequence FBNY (127 bp) and a sex-independent control PCR-fragment, the microsatellite marker FBN17 (136-140 bp) are generated in the same PCR reaction. Synteny mapping assigned the male-specific sequence to bovine chromosome Y (BTA Y), whereas FBN17 was mapped to bovine chromosome 2. Localisation of FBNY on BTA Y was confirmed by fluorescence in hybridisation of two BAC clones containing the male-specific sequence. There was no amplification of the male-specific target sequence FBNY in sheep, pig, goat, mice, man, and several wild species of the tribe Bovini. The bovine male-specific fragment was detected in dilutions containing as little as 10 pg genomic DNA and in blastomeres from embryo biopsies. The PCR assay presented here does require neither restriction endonuclease digestion of the PCR product nor additional nested PCR steps. Owing to the advantage of parallel amplification of the autosomal locus FBN17 no additional control fragment is necessary to detect PCR failure. The results of sex determination in embryo biopsies using FBNY were in agreement with the outcome from a reference assay used in commercial breeding programs.

Animals↗
PubMed1998

Molecular cloning and complete nucleotide sequence of galinsoga mosaic virus genomic RNA.

The complete genomic sequence of galinsoga mosaic virus (GaMV) was determined. The genome consists of 3,803 nucleotides and has five open reading frames (ORFs). The 5' ORF (ORF 1) encodes a protein with predicted molecular mass of 23 kDa and readthrough of its amber stop codon probably yields a 82 kDa protein (ORF 2). ORFs 3 and 4 encode two polypeptides with molecular masses of 8 and 7 kDa, respectively. ORF 5 encodes the 36 kDa capsid protein. Amino acid sequence comparisons revealed that the nonstructural proteins encoded by ORFs 1, 3, and 4 were more similar to the corresponding gene products of tobacco necrosis virus, strain A, than to those of carmoviruses. Conversely, the coat protein was more similar to that of tombusviruses. The readthrough region of the viral replicase (ORF 2) had high sequence homology with that of carmo-, tombus-, and necroviruses. Computer analysis of the protein encoded by ORF 1 as well as of the corresponding product of turnip crinkle (TCV) and melon necrotic spot (MNSV) carmoviruses revealed the presence of a sequence with local hydrophobicity and hydrophobic moment characteristic of mitochondrial targeting sequence which may explain the origin of the carmovirus-induced multivesicular bodies from mitochondria.

Amino Acid Sequence↗
PubMed1994

Differences in in vivo and in vitro sequence-specific sites of cisplatin-DNA adduct formation and detection of a dose-response relationship.

The cytotoxic and mutagenic properties of the anticancer drug cis-diammine-dichloroplatinum(II) (cisplatin) are mediated by bifunctional adducts between purines. Experiments performed in this study employed a new repetitive thermal-cycling technique to detect cisplatin adduct formation following exposure of cells in culture (in vivo) or following treatment of purified DNA (in vitro exposure). The initial goal of this study was to determine if cisplatin-DNA adduct formation could be measured accurately using phosphor-imaging over a broad concentration range. If this proved possible, it would then be feasible to determine if adduct formation differed within chromatin compared with purified DNA. There were no significant differences in the cisplatin-DNA adduct pattern induced in closed circular or linear double-stranded plasmids in vitro, suggesting that this type of tertiary structural change does not affect the formation of adduct sites. Sequence-specific DNA adduct formation within a human repetitive DNA target sequence, alphoid DNA, following cisplatin treatment of prostate cancer cells in culture (in vivo) and treatment of purified DNA in vitro revealed consistent increases in adduct formation over a broad concentration range, validating the experimental technique. Comparing preferences for cisplatin adduct site formation under these different conditions of exposure demonstrated statistically significant differences. Similar differences were detected for cisplatin repair-deficient Xeroderma pigmentosum cells treated in cell culture, indicating that in vivo/in vitro preferences for adduct site formation are not the result of DNA repair in vivo.

Base Sequence↗
PubMed1992

Interaction of singlet oxygen with DNA and biological consequences.

To study the interaction of singlet oxygen (1O2) with DNA and the biological consequences of 1O2-induced DNA damage, we used the thermodissociable endoperoxide of 3,3'-(1,4 naphthalidene) dipropionate (NDPO2) as a generator of free 1O2 in reactions with (1) 2'-deoxynucleoside 3'-monophosphates (dNps), (2) an oligonucleotide (16-mer) having one deoxyguanine (dG), (3) native and denaturated rat kidney DNA and (4) single-stranded (ss) and double-stranded (ds) bacteriophage M13mp10 DNA. Using both anion exchange and reversed phase HPLC and 32P-postlabeling analyses, it was found that exposure of the various dNps to chemically generated 1O2 led to a detectable reaction with dGp and not with dAp, dCp, d5mCp or Tp. The reaction with dGp led to degradation of this nucleotide and the formation of a large number of reaction products, one of which could be identified as 7-hydro-8-oxo-2'-deoxyguanosine 3'-monophosphate (8-oxo-dGp). A second product could tentatively be identified as a formamido pyrimidine derivative of dGp (Fapy-dGp). When ss DNA, ds DNA or the oligonucleotide were exposed to 1O2, the formation of 8-oxo-dG could also be demonstrated. With the oligonucleotide, we found a so far unidentified reaction product. Under the same reaction conditions the yield of 8-oxo-dG was about 8-fold higher in ss DNA than in ds DNA. In ss DNA 8-oxo-dG seemed to be a more prominent product than in the case of reaction of 1O2 with free dGp. Reaction of 1O2 with ss or ds M13mp10 DNA led to biological inactivation of these DNAs, ss DNA being at least 100-fold more sensitive than ds DNA. It could be concluded that inactivation of the ss DNA must be largely due to 1O2-induced DNA lesions other than 8-oxo-dG. In agreement with the observed preferential reaction of 1O2 with dG most of the so far sequenced mutations, induced by 1O2 in a 144 bp mutation target sequence inserted in the lacZ alpha gene of ss or ds M13mp10 DNA, occurred at a G or G/C base pair respectively. A preference for G(C) to T(A) transversions can be observed for which 8-oxo-dG might have been responsible. In ss DNA a significant number of the mutations are characterized by the fact that a G is deleted.

Animals↗
PubMed2001

Quantification of ovine cytokine gene expression by a competitive RT-PCR method.

A quantitative competitive RT-PCR method was developed in order to measure IL-1beta, IL-4, IL-12, IFNgamma, TNFalpha and G(3)PDH mRNA from samples of ovine tissue such as lymph node or spleen. The main advantage of the method relies on the use, for each target sequence, of an internal competitor construct similar to the relevant target, but 4-bp different in size. This competitive strategy is validated by the equivalence of the amplification process, observed separately between competitor DNA and target DNA species. Furthermore, the copy number of each cytokine cDNA is normalized to a fixed copy number of G(3)PDH cDNA. The cDNA level of this constitutive gene was effectively shown to remain constant whatever the tissue studied and independently of the experimental conditions used. The accurate and reproducible data obtained permit the application of this quantitative RT-PCR method to measure the sheep cytokine response to Salmonella infection. Early induction of IFNgamma mRNA was observed in the draining lymph node 1 day after infection. At the same time, a strong increase of IL-1beta mRNA was observed in local and systemic lymphoid organs, suggesting the initiation of the inflammatory response. Finally, the overall results demonstrate the efficiency of the method and its suitability for further studies of the immune response in the ovine species.

Animals↗