Foreign Promoter Research Articles

Interactions of Drosophila Ultrabithorax regulatory regions with native and foreign promoters.

The Ultrabithorax (Ubx) gene of the Drosophila bithorax complex is required to specify parasegments 5 and 6. Two P-element "enhancer traps" have been recovered within the locus that contain the bacterial lacZ gene under the control of the P-element promoter. The P insertion that is closer to the Ubx promoter expresses lacZ in a pattern similar to that of the normal Ubx gene, but also in parasegment 4 during embryonic development. Two deletions have been recovered that remove the normal Ubx promoter plus several kilobases on either side, but retain the lacZ reporter gene. The lacZ patterns from the deletion derivatives closely match the normal pattern of Ubx expression in late embryos and imaginal discs. The lacZ genes in the deletion derivatives are also negatively regulated by Ubx and activated in trans by Contrabithorax mutations, again like the normal Ubx gene. Thus, the deleted regions, including several kilobases around the Ubx promoter, are not required for long range interactions with Ubx regulatory regions. The deletion derivatives also stimulate transvection, a pairing-dependent interaction with the Ubx promoter on the homologous chromosome.

Long-term expression of the hepatitis B virus core-e- and X-proteins does not cause pathologic changes in transgenic mice

Background/Aims: Chronic infections with the human hepatitis B virus can result in liver cirrhosis and primary hepatocellular carcinoma. The reasons for these long-term effects are unclear. The aim of this study was to generate transgenic mice expressing the HBV X- and c/e-gene under authentic and foreign promoter control and to test whether the respective gene products can cause pathologic effects during the lifespan of a mouse. Moreover, the temporal and the tissue-specific regulation of the crucial HBV c/e-gene promoter was analyzed. Methods: Eight transgenic mouse lines were generated. Four contained the c/e- and X-gene and two contained only the X-gene under authentic promoter control. Two lines expressed only the X-gene under control of the rat insulin promoter/enhancer. Gene expression was tested by protein and mRNA analyses. During an observation period of 2 years, mice were sacrificed and organs subjected to histologic examination. Mice expressing the X-gene in pancreatic beta cells were tested for the development of diabetes. Results: In the liver, slight histopathologic alterations but no neoplastic changes could be observed in mice expressing the X-gene. Activity of the c/e-gene promoter/enhancer was age dependent and was not restricted to hepatocytes. Conclusion: No evidence was obtained that long-term expression of the HBV c/e- and X-gene products can cause neoplasia during the lifespan of a mouse.

Species-specificity of rRNA gene transcription in plants manifested as a switch in RNA polymerase specificity.

Rapid evolution of ribosomal RNA (rRNA) gene promoters often prevents their recognition in a foreign species. Unlike animal systems, we show that foreign plant rRNA gene promoters are recognized in an alien species, but tend to program transcription by a different polymerase. In plants, RNA polymerase I transcripts initiate at a TATATA element (+1 is underlined) important for promoter strength and start-site selection. However, transcripts initiate from +32 following transfection of a tomato promoter into Arabidopsis. The rRNA gene promoter of a more closely related species, Brassica oleracea, programs both +1 and +29 transcription. A point mutation at +2 improving the identity between the Brassica and Arabidopsis promoters increases +1 transcription, indicating a role for the initiator element in species-specificity. Brassica +29 transcripts can be translated to express a luciferase reporter gene, implicating RNA polymerase II. TATA mutations that disrupt TATA-binding protein (TBP) interactions inhibit +29 transcription and luciferase expression. Co-expressed TBP proteins bearing compensatory mutations restore +29 transcription and luciferase activity, suggesting a direct TBP-TATA interaction. Importantly, +1 transcription is unaffected by the TATA mutations, suggesting that in the context of pol I recognition, the TATA-containing initiator element serves a function other than TBP binding.

Tetrahydrobiopterin-dependent Production of L-DOPA in Genetically Modified Primary Fibroblasts: Basic Research of Gene Therapy for Parkinson's Disease

Summary Although intracerebral transplantation might become a new strategy for the treatment of neurological diseases, there are many problems for the grafts, We examined the possibilities of primary skin fibroblasts as a new graft for Parkinson's disease. Rat primary skin fibroblasts were transfected with a retrovirus vector containing eDNA of human tyrosine hydroxylast typel (TH) (pLTHSNL) or cytomegalovirus promoter (pCTHSNL) as a foreign promoter. In these genetically modified fibroblasts, catecholamine production and release were analyzed in vitro by immunocytochemistry and high performance liquid chromatography with electrochemical detection (HPLC-ECD), Being supplemented with biopterin (BH4; (6R)-L-erythro-tetrahydrobiopterin) cofactors required for TH activity, these cells produced and released L-DOPA into the culture medium. When combined with a foreign promoter and BH4, L-DOPA production increased in a timedependent manner, which was not affected by the number of cell-passages and the duration of liquid nitrogen freezing . These facts suggest that the amount of L-DOPA secreted from transplanted genetically modified fibroblasts can be manipulated by an inserted promoter the exogenously administered BH4. Thus genetically modified primary skin fibroblasts transfected with THcDNA using retrovirus vector system may hold promise as a graft for transplantation and gene therapy for Parkinson's disease.

Replication-defective herpes simplex virus vectors for gene transfer in vivo.

Herpes simplex virus 1 has a number of biological features which suggest that it could be engineered as a vector for direct transfer of therapeutic genes to neurons. These features include (i) its natural ability to establish life-long latency, a state in which the viral genome is not integrated, lytic genes are quiescent, and the metabolic functioning of the host cell is apparently undisturbed; (ii) the expression of latencyassociated transcripts (LATs) driven by neuron-specific, latency-active promoter (LAP) elements, which may prove useful in expressing transgenes from latent viral genomes; and (iii) the observation that replication-defective mutants created by the deletion of essential genes retain the ability to establish a latent state in the nervous system (1). In addition, many of the 81 herpes simplex virus (HSV) genes are not required for viral replication in cell culture and may conveniently be deleted to provide space for incorporation of substantial foreign DNA, and almost all viral genes are contiguous units, making genetic manipulation feasible. The virus can also be grown to high titer, and viral infectivity is very efficient. The major impediments to the development of HSV-effective vectors relate to residual cytotoxicity of defective vectors and the limited duration of transgene expression. Even replication-incompetent mutant viruses are cytotoxic, readily killing neurons in vitro, and with the exception of the HSV LAP elements, viral and foreign promoters appear to come under control of the virus' ability to rapidly induce mechanisms of promoter shutoff. Two different types of HSV-based gene delivery systems have been developed. The type consists of genetically engineered genomic vectors, which may be deleted in genes required for the virus to replicate in postmitotic cells such as neurons or may be completely replication-defective, requiring complementation for vector propagation. The second type of HSV-based vector system, referred to as amplicons, uses defective helper-virus mutants for packaging concatemeric plasmids containing an HSV origin of DNA synthesis and a packaging sequence. We have focused our efforts on the development of replication-defective genomic vectors. The first generation defective genomic vectors were deleted in the single essential immediate early (IE) gene encoding ICP4 (e.g., d120) (2). These vectors can be propagated in ICP4-complementing cell lines, but on infection of neurons, viral gene expression is aborted at the level of IE gene expression. Although these vectors are of reduced pathogenicity and can be used to efficiently transfer and transiently express reporter genes in brain (see below), they are toxic to neurons in culture, producing cytopathic effects such as cytoplasmic blebbing, host cell DNA fragmentation, and chromo-

Construction of derivatives of the F plasmid pOX-tra715: characterization of traY and traD mutants that can be complemented in trans.

A derivative of the F plasmid, pOX38-tra715, expresses the entire F tra operon from a foreign promoter (PT7) derived from phage T7. A series of plasmids related to pOX38-tra715 were constructed which carry either deletion mutations or point mutations in traY. When the PT7 promoter was induced, these plasmids expressed the F pilus but were transfer deficient unless TraY was supplied In trans from compatible plasmids. Insertion of a kanamycin-resistance cassette in the traY gene of the pOX38 plasmid, which contains the wild-type PY promoter, resulted in loss of F piliation and transfer ability. Introduction of TraY in trans partially restored piliation and transfer suggesting that TraY has a role in positively regulating the PY promoter, pOX38-tra719-traD411, which contains a chloramphenicol-resistance cassette in place of the kanamycin-resistance cassette in pOX38-tra715 and a mutation in traD, was constructed to demonstrate the utility of this series of plasmids in studying the long (30 kb) F tra operon.

Regulation of L-DOPA production by genetically modified primary fibroblasts transfected with retrovirus vector system

Although intracerebral grafting has become established as a new strategy for the treatment of Parkinson's disease, there are many problems regarding such grafts. We focused on the grafting of primary skin fibroblasts. Rat primary skin fibroblasts were transfected with a retrovirus vector containing cDNA of human tyrosine hydroxylase (TH) type 1 (LTHSNL) or of cytomegalovirus promoter (CTHSNL) as a foreign promoter. In these genetically modified fibroblasts, L-DOPA production and release were analyzed in vitro by immunocytochemistry and high-performance liquid chromatography with electrochemical detection (HPLC-ECD). Being supplemented with the biopterin (BH4:(6R)-L-erythro-tetrahydrobiopterin) cofactors required for TH activity, these cells produced and released L-DOPA into the culture medium. When CTHSNL and BH4 were combined, L-DOPA production increased with time, regardless of the number of cell passages, or the duration of liquid nitrogen freezing. This suggests that the foreign gene (THcDNA) containing retrovirus vector integrates into the chromosomal DNA of the target cells (fibroblasts). Primary fibroblasts can be easily obtained and cultured. Thus, genetically modified primary skin fibroblasts transfected with a retrovirus vector system containing the TH cDNA may be promising grafts for transplantation and gene therapy in Parkinson's disease.

Regulation of Na+/H+ Exchanger Gene Expression: ROLE OF A NOVEL POLY(dA·dT) ELEMENT IN REGULATION OF THE NHE1 PROMOTER

In this study we examine regulation of expression of the Na+/H+ exchanger promoter in L6 and NIH 3T3 cells. We have identified a highly conserved poly(dA dT)-rich region that appears to be important in regulation of expression of the NHE1 gene. Deletion or mutation of this region results in dramatic decreases in promoter activity in both L6 and NIH 3T3 cells. In addition, DNase I footprinting experiments demonstrated that this region is protected by nuclear extracts from both cell types, and gel mobility shift assays showed that a protein or proteins specifically binds to the poly(dA dT)-rich element. Using Southwestern blotting, we determined that a 33-kDa protein binds to the poly(dA dT)-containing region. Mutations that abolished protein binding to this element diminished activity of the promoter. Insertion of the poly(dA dT)-rich element into a plasmid containing the SV40 promoter demonstrated that this element can also enhance the activity of a foreign promoter. Together, the results we have presented here show that the poly(dA dT)-rich region is important in regulation of NHE1 expression in different cell types.

On the Role of Helix 0 of the Tryptophan Synthetase α Chain of Escherichia coli

The role of helix 0 of the alpha chain (TrpA) of the tryptophan synthetase alpha2beta2 multi-functional enzyme complex of Escherichia coli was examined by deleting amino-terminal residues 2-6, 2-11, or 2-19 of TrpA. Selected substitutions were also introduced at TrpA positions 2-6. The altered genes encoding these polypeptides were overexpressed from a foreign promoter on a multicopy plasmid and following insertion at their normal chromosomal location. Each deletion polypeptide was functional in vivo. However all appeared to be somewhat more labile and insoluble and less active enzymatically than wild type TrpA. The deletion polypeptides were overproduced and solubilized from cell debris by denaturation and refolding. Several were partially purified and assayed in various reactions in the presence of tryptophan synthetase beta2 (TrpB). The purified TrpADelta2-6 and TrpADelta2-11 deletion polypeptides had low activity in both the indole + serine --> tryptophan reaction and the indoleglycerol phosphate + serine --> tryptophan reaction. Poor activity in each reaction was partly due to reduced association of TrpA with TrpB. The addition of the TrpA ligands, alpha-glycerophosphate or indoleglycerol phosphate, during catalysis of the indole + serine --> tryptophan reaction increased association and activity. These findings suggest that removal of helix 0 of TrpA decreases TrpA-TrpB association as well as the activity of the TrpA active site. Alignment of the TrpA sequences from different species indicates that several lack part or all of helix 0. In some of these polypeptides, extra residues at the carboxyl end may substitute for helix 0.

The centromere-like parC locus of plasmid R1.

The parA partitioning system of plasmid R1 consists of three components: the cis-acting centromere-like parC locus, and two proteins, ParM and ParR. The parC locus contains two sets of five direct repeats (iterons) to which the ParR protein binds. The parA promoter is located in the core region between the two sets of iterons. Mini-R1 replicons carrying parC are stabilized by the simultaneous presence of ParM and ParR. The parC locus present on a co-resident plasmid leads to instability of the mini-R1 replicon (incompatibility). Here we present a genetic analysis of the stability and incompatibility phenotypes associated with parC. We show that all 10 iterons are required for maximum stabilization and incompatibility. Replacement of the core promoter region between the repeats by a foreign promoter region did not reduce stabilization. Thus, the only structural components in parC seem to be the two sets of iterons. The parA promoter, P parA, is repressed by ParR. We show that all 10 iterons are required for full repression of the promoter. The activity of the promoter was influenced by sequences located outside the core region. An A-rich region located upstream of the -35 element of PparA was found to increase promoter activity. The region encoding the parA mRNA leader region also strongly influenced the expression level of PparA- lacZ fusions. We show that this high expression (hex) element is a transcriptional antiterminator that prevents Rho-dependent termination.

The HBV-Producing Cell Line HepG2-4A5: A Newin VitroSystem for Studying the Regulation of HBV Replication and for Screening Anti-Hepatitis B Virus Drugs

All tissue culture systems for propagating HBV employed so far make use of tandemly arranged HBV genomes usually under the control of strong foreign promoters. Thus these systems are helpful for virus production but are of limited value in the investigation of the regulation of HBV replication or of the extent to which the expression of viral genes might be influenced by cellular signal transduction pathways. To overcome this barrier we established an HBV-producing cell line (HepG2-4A5) by stably transfecting HepG2 cells with a replication-competent, terminally redundant HBV plasmid (pSPT1.2 ×HBV) that contains each of the four major HBV-ORFs only once and exclusively under the control of their own regulatory elements. HepG2-4A5 cells contain a single, nonrearranged, chromosomally integrated, replication-competent HBV genome. In the cytoplasm of HepG2-4A5 cells, all typical viral mRNAs were detectable, but no other viral transcripts were found. Furthermore, all viral gene products are synthesized in a balanced ratio, as close as possible to that found in anin vivoinfection. Dane-like particles released from HepG2-4A5 cells were indistinguishable from virions synthesizedin vivo,by all physical (electron microscopy, buoyant density) and biochemical (endogenous polymerase reaction, immunogenic behaviour) criteria. Because of the autologous genome organization in this system, the HepG2-4A5 cell line allows studies on the function of the HBV gene products with respect to their involvement in regulating HBV replication under conditions imitating as closely as possible the situationin vivo.Furthermore, this cell line might be a helpful tool in screening antiviral drugs and in studying their effect on regulating HBV replication.

Strong expression of peroxisomal malate synthase genes from n-alkane-utilizable yeast Candida tropicalis in Saccharomyces cerevisiae

Expression of the genes encoding several peroxisomal enzymes of the n-alkane-utilizable yeast Candida tropicalis has been examined in Saccharomyces cerevisiae to find out a novel and foreign promoter functional in S. cerevisiae. Among these genes, two malate synthase genes, as well as the isocitrate lyase gene, were strongly expressed in acetate-grown cells of S. cerevisiae. The amounts of the recombinant malate synthases expressed in S. cerevisiae were as much as 30% of the total amount of soluble proteins in the cells. These results showed that the upstream regions (1.1 kbp for the MS-1 gene and 0.8 kbp for the MS-2 gene) of the malate synthase genes [ UPR-MSs] from C. tropicalis contained strong promoters functional in S. cerevisiae and that the construction of novel gene expression systems using UPR-MSs might be possible.

Requirements for Optimal Expression of Secreted and Nonsecreted Recombinant Proteins in Vaccinia Virus Systems

Selection of an optimal promoter is necessary for efficient expression of foreign genes with vaccinia virus. Since a variety of powerful (homologous) vaccinia virus promoters and foreign (heterologous) promoter systems have been described for use in vaccinia, we have addressed the question of whether a general rule exists that allows the prediction of the optimal promoter/gene combination. We have compared the expression properties of four secreted proteins, the human blood clotting factor IX (FM), the human blood glycoprotein Protein S (ProtS), the human von Willebrand factor (vWF), and the Hepatitis B virus (HBV) middle surface glycoprotein preS2, with proteins that were reported not to be secreted, the HBV large surface glycoprotein preS1 and the murine leukemia virus (MuLV) BM-5 Eco gag protein. In addition, we have included in our study an internal control protein, the vaccinia virus pll protein, to monitor possible side effects of the promoter system used. Genes encoding the foreign proteins were placed either under control of a synthetic vaccinia virus early/late promoter (selP) or under control of the bacteriophage T7 promoter (T7/emc system). The secreted proteins were more efficiently expressed when fused to the homologous promoter. Direct comparison of the two promoters indicated that the expression level ranged between 1.4 (ProtS) and 3.9 (FM)-fold higher with the selP than with the T7 promoter. In contrast, the cell-associated HBV preS1 was more efficiently expressed under the T7 promoter and the MuLV BM-5 Eco gag polypeptide was expressed equally well from both promoters. These data indicate that a careful prediction of optimal promoter/foreign gene combinations for the vaccinia virus expression system is possible. The choice of the optimal promoter/expression system is based on a simple classification scheme, discriminating secreted and nonsecreted proteins.

A second N-acylhomoserine lactone signal produced by Pseudomonas aeruginosa.

Quorum sensing systems are used by a number of Gram-negative bacterial species to regulate specific sets of genes in a cell density-dependent manner. Quorum sensing involves synthesis and detection of extracellular signals termed autoinducers. As shown in recombinant Escherichia coli, the Pseudomonas aeruginosa autoinducer (PAI) N-(3-oxododecanoyl)homoserine lactone, together with the lasR gene product, activate the P. aeruginosa lasB gene. In this study, PAI was shown to activate lasB-lacZ expression in a P. aeruginosa lasR mutant containing a plasmid with lasR under the control of the lac promoter. The concentration of PAI necessary for half-maximal activation of the lasB-lacZ fusion was approximately 1 microM, which is within the range of PAI levels found in P. aeruginosa culture fluids. The effect of PAI on a P. aeruginosa lasR mutant containing a plasmid with lasR under the control of its own promoter and containing the lasB-lacZ fusion was also tested. Although extracts of culture fluid activated the lasB promoter in this construct, concentrations of PAI as high as 10 microM did not. This indicates the presence of a second extracellular factor (factor 2) that is required for lasB activation in P. aeruginosa when lasR is controlled by its own promoter but not when lasR is controlled by a strong foreign promoter. Factor 2 was shown to be N-butyrylhomoserine lactone. Although recombinant E. coli cells containing the PAI synthase gene, lasI, produce PAI, these cells do not produce factor 2. Furthermore, a P. aeruginosa mutant that produced about 0.1% of the wild-type level of PAI made about 5% of the wild-type level of factor 2. This indicates that factor 2 synthesis results from the activity of a gene product other than PAI synthase. The role of factor 2 in virulence gene regulation remains to be determined, but this compound may affect the expression of lasR, which in turn activates transcription of numerous virulence genes in the presence of sufficient PAI. Apparently, multiple quorum sensing systems can occur and interact with each other in a single bacterial species.

An early experiment in the reorganisation of agricultural production in the French Soudan (Mali), 1920–40

Water control has long appeared an attractive technological solution to risky farming i n zones dependent on rain-fed cropping systems, especially in semi-arid regions. From the early twentieth century, European technicians and administrators sought to develop irrigated agriculture in African colonies. In the French Soudan the earliest colonial waterworks date back to the 1920s, just outside Bamako, in the vicinity of Baguineda. From Baguineda the French went on to develop a much larger-scale irrigation project north of Segou known as the Office du Niger. This study uses archival documents to show that a new system of crop production was imposed on peasants by the colonial state. Using labour requisitions and in some cases forced resettlement, the colony introduced new crops and technology for rapid intensification, and in so doing organised a new agricultural system oriented primarily towards the market. The Baguineda project began as, and remained, an enclave, spatially distinct from the surrounding dryland grain and pulse cropping system oriented primarily towards lineage reproduction. In common with many subsequent introduced agricultural development projects in Africa, its lack of success was due in part to the conflicting interests of poor peasant workers and powerful foreign promoters.

A sensitive promoter assay based on the transcriptional activator Tat of the HIV-1 virus

We developed a sensitive vector system for the analysis of weak promoter activities. This promoter assay is based on the transcriptional activator protein. Tat, of human immunodeficiency virus type 1 (HIV-1). High-level expression of HIV requires activation in trans by Tat of the promoter in the long terminal repeat (LTR). Here we describe the construction of a promoterless pTat vector. Foreign promoter elements can be inserted upstream from the tat gene, and expression of Tat protein is measured in trans on a co-transfected LTR-CAT reporter plasmid. We show that this binary system is more sensitive than standard pCAT reporter assays.

Growth factor-mediated induction of the delayed early gene T1 depends on a 12-O-tetradecanoylphorbol 13-acetate-responsive element located 3.6 kb upstream of the transcription initiation site.

The T1 gene is a delayed early serum-responsive gene which encodes a secreted glycoprotein of the immunoglobulin superfamily. We have addressed the question of what promoter elements are needed to allow for growth factor-mediated T1 gene expression. By deletion analysis we have identified a 448-bp DNA region 3.5-4.0 kb upstream of the transcription start site which can confer serum inducibility onto a foreign minimal promoter. Within this sequence there is a 12-O-tetradecanoylphorbol 13-acetate (TPA)-responsive element (TRE) which is essential for T1 promoter induction in response to the forced expression of the transcription factor AP-1 in NIH 3T3 fibroblasts and F9 teratocarcinoma cells. This TRE is crucial for growth factor-mediated T1 gene expression. A point mutation within this TRE attenuated serum inducibility. Two E boxes are positioned 6 and 40 bp downstream of the TRE. Point mutations within these sequence motifs reduced basal T1 promoter activity and serum inducibility. Additional, as-yet-unidentified, promoter elements within the 448-bp serum-responsive region are required for T1 gene activation in response to growth stimulation.

Characterization of HCV structural proteins expressed in various animal cells.

Hepatitis C virus (HCV) is a main causative agent for transfusion-associated and sporadic cases of non-A, non-B hepatitis throughout the world. HCV has a positive-strand RNA of about 9,400 nucleotides as its genome, whose organization is similar to those of animal pestiviruses or human flaviviruses. In spite of the lack of an effective replication system in tissue culture cells, genes coding for viral proteins of HCV have been identified. The putative nucleocapsid (p22) and envelope (gp35 and gp60) proteins have been expressed in cells by different vectors under various foreign promoters. Furthermore, a truncated core protein and association of envelope proteins with nonstructural proteins have also been observed. These synthesized viral proteins have been shown to be useful for diagnostic assays.

A novel response-regulator is able to suppress the nodulation defect of a Bradyrhizobium japonicum nodW mutant

The two-component regulatory system Nod-VW of Bradyrhizobium japonicum is essential for the nodulation of the legume host plants Vigna radiata, V. unguiculata and Macroptilium atropurpureum. The NodV protein shares homology with the sensor-kinases, whereas the NodW protein is a member of the response-regulator class. We report here the identification of a new B. japonicum DNA region that is able to suppress the phenotypic defect of a nodW mutant, provided that this region is expressed from a foreign promoter. The minimal complementing region, which itself is not essential for nodulation in a nodW+ background, consists of one gene designated nwsB (nodW-suppressor). The deduced amino acid sequence of the nwsB gene product shows a high degree of homology to NodW. The nws B gene is preceded by a long open reading frame, nwsA, whose putative product appears to be a sensor-kinase. Downstream of nwsB, an open reading frame encoding a second putative response-regulator was identified. Interspecies hybridization revealed the presence of nwsAB-like DNA also in other Bradyrhizobium strains. Using nwsB'-'lacZ fusions, the nwsB gene was found to be expressed rather weakly in B. japonicum. This low level of expression is obviously not sufficient to compensate for a nodW- defect, whereas strong overexpression of nwsB is a condition that leads to suppression of the nodW- mutation.

The molecular biology of hepatitis C virus

Hepatitis C virus (HCV) is one of the main causative agents for transfusion associated- and sporadic cases of non-A, non-B hepatitis throughout the world. HCV has a positive strand RNA of about 9400 nucleotides, as its genome, whose organization is similar to those of animal pestiviruses or human flaviviruses. In spite of the lack of the effective replication system in tissue culture cells, parts of the viral genome were expressed under the control of foreign promoters and the synthesized viral protein has been used for diagnostic assays.