Partial Genomic Clone Research Articles

Molecular cloning and characterization of cDNAs of the superoxide dismutase gene family in the resurrection plant Haberlea rhodopensis

Resurrection plants can tolerate almost complete water loss in their vegetative parts. The superoxide dismutases (SODs) are essential enzymes of defense against the oxidative damage caused by water stress. Here, we cloned and characterized cDNAs of the SOD gene family in the resurrection plant Haberlea rhodopensis. Seven full-length cDNAs, and their partial genomic clones, were obtained by combination of degenerate PCR, RT-PCR and RACE. The derived amino acid sequences exhibited a very high degree of similarity to cytosolic Cu,Zn-SODs (HrCSD2, HrCSD3), chloroplastic Cu,Zn-SODs (HrCSD5), other Cu,Zn-SODs (HrCSD4), Mn-SODs (HrMSD) and Fe-SODs (HrFSD). One cDNA turned out to be a pseudogene (HrCSD1). All identified SOD genes were found expressed at transcriptional level--the HrCSD2, HrCSD5, HrMSD and HrFSD were constitutively expressed in all organs, while the HrCSD3 and HrCSD4 were organ-specific. The transcripts of the housekeeping SOD genes were detected at significant levels even in air-dry leaves. The multigene SOD family of H. rhodopensis is the first studied SOD family amongst resurrection plant species. Our finding of well expressed SOD transcripts in fully dehydrated leaves correlates with retention of SOD activity, and with the ability of H. rhodopensis to revive upon rehydration. Because of the endemic relict nature of that species, our findings may help to further elucidate the evolutionary relationships among different SOD isoforms from distinct plant species.

The vitronectin gene in rainbow trout: Cloning, expression and phylogenetic analysis

Vitronectin is a major cell adhesion glycoprotein that is found in plasma and the extracellular matrix. Vitronectin consists of an N-terminal somatomedin B domain and two hemopexin-like domains and controls functions including cell adhesion, migration, haemostasis and immune defence. In order to study the molecular evolution of the complement lytic pathway regulation, we have cloned and characterized the vitronectin gene from rainbow trout ( Oncorhynchus mykiss). The deduced amino acid sequence of trout vitronectin exhibits 45%, 46%, 47% and 63% identity with human, chicken, Xenopus and zebrafish orthologs, respectively. The domain architecture of the trout vitronectin, consisting of a somatomedin B domain and two hemopexin-like domains, resembles that of mammalian vitronectins. Analysis of partial genomic clones shows that trout vitronectin gene exhibits the same exon–intron organization profile as the human ortholog gene. The trout vitronectin gene is probably present as a single copy in the trout genome, showing a differential expression pattern among tissues investigated.

Fiber-FISH analysis of the 3'-terminal region of the human L-type Ca2+ channel alpha 1C subunit gene.

Human L-type Ca2+ channel alpha 1C subunit gene (CACNL1A1) maps to the distal region of chromosome 12p13, and is composed of approximately 50 exons spanning over 150 kb of the human genome as estimated by restriction map analysis. However, the structure and the total length of the 3'-end of the gene is not clear because the size of several big introns remains unknown. Here the fiber-FISH technique was used to determine the relative order and size of eight partial genomic DNA clones from the central and 3'-terminal regions of CACNL1A1. The total physical distance of this region, including the size and gap distances between the clones were re-estimated. The results show that the physical order of the tested clones was 5'-g14-5 > g12-2 > g10-8 > g4-5 > g16-7 > g8-3 > g12-5 > g6-20-3'. Their individual sizes vary between 6.7 and 21.9 kb. Clones g6-20 and g12-5, both containing repetitive exon 45/46-like element, were found to be located within 59.1 kb downstream of g8-3 containing earlier identified polyadenylation site, i.e. 229.5 kb away from clone g14-5 (exons 10, 11). The possible implications of this structural complexity is discussed.

Expression pattern of Cubitus interruptus from the mulberry silkworm Bombyx mori in late developmental stages.

A partial genomic clone of Bombyx mori homologue of the segment polarity gene Cubitus interruptus ( BmCi), encoding the conserved zinc finger domain and harbouring two introns, has been characterized. BmCi was expressed in the silkglands of B. mori from embryonic to the late larval stages(3rd, 4th and 5th intermoults). The expression was confined to the anterior region of the middle silkglands, overlapping with the domain of sericin-2 expression and excluding the domains of Bm invected expression, namely the middle and posterior regions of the middle silkglands. In the wing discs, the expression was restricted to the anterior compartment, which increased from 4th to 5th larval intermoults and declined later in the pupal wing buds. In gonadal tissues (both ovaries and testes) BmCi was expressed from the larval to pupal stages. The transcripts were localized to the sperm tubes containing spermatogonia in the testis of Bombyx larvae. BmCi expression, however, was not detected in any of these tissues during the moulting stages. Expression of Ci in the wing discs and gonads is evolutionarily conserved, while the silkgland represents a novel domain. Our results imply that BmCi is involved in the specification and maintenance of micro-compartment identity within the middle silkglands.

Isolation and characterization of a cDNA encoding a lipid transfer protein expressed in 'Valencia' orange during abscission.

The genetics and expression of a lipid transfer protein (LTP) gene was examined during abscission of mature fruit of 'Valencia' orange. A cDNA encoding an LTP, CsLTP, was isolated from a cDNA subtraction library constructed from mature fruit abscission zones 48 h after application of a mature fruit-specific abscission agent, 5-chloro-3-methyl-4-nitro-pyrazole (CMN-pyrazole). A full-length cDNA clone of 652 nucleotides was isolated using 5' and 3' RACE followed by cDNA library screening and PCR amplification. The cDNA clone encoded a protein of 155 amino acid residues with a molecular mass and isoelectric point of 9.18 kDa and 9.12, respectively. A partial genomic clone of 505 nucleotides containing one intron of 101 base pairs was amplified from leaf genomic DNA. Southern blot hybridization demonstrated that at least two closely related CsLTP genes are present in 'Valencia' orange. Temporal expression patterns in mature fruit abscission zones were examined by northern hybridization. Increased expression of CsLTP mRNA was detected in RNA of mature fruit abscission zones 6, 24, 48, and 72 h after application of a non-specific abscission agent, ethephon. Low expression of CsLTP transcripts was observed after treatment of CMN-pyrazole until 24 h after application. After this time, expression markedly increased. The results suggest that CsLTP has a role in the abscission process, possibly by assisting transport of cutin monomers to the fracture plane of the abscission zone or through its anti-microbial activity by reducing the potential of microbial attack.

R9AP, a membrane anchor for the photoreceptor GTPase accelerating protein, RGS9-1.

The regulator of G protein signaling (RGS)-9-1.G(beta 5) complex forms the GTPase accelerating protein for G(alpha t) in vertebrate photoreceptors. Although the complex is soluble when expressed in vitro, extraction of the endogenous protein from membranes requires detergents. The detergent extracts contain a complex of RGS9-1, G(beta 5), G(alpha t), and a 25-kDa phosphoprotein, R9AP (RGS9-1-Anchor Protein). R9AP is encoded by one intronless gene in both human and mouse. Full or partial cDNA or genomic clones were obtained from mice, cattle, human, zebrafish, and Xenopus laevis. R9AP mRNA was detected only in the retina, and the protein only in photoreceptors. R9AP binds to the N-terminal domain of RGS9-1, and anchors it to the disk membrane via a C-terminal transmembrane helix.

Brachyury (T) gene expression and notochord development in Oikopleura longicauda (Appendicularia, Urochordata).

Appendicularia (Larvacea) is a subgroup of Urochordata (Tunicata) comprised of holoplanktonic organisms that retain their tailed architecture throughout their life history, while other tunicates, including ascidians and doliolids, resorb the tail after metamorphosis. In order to investigate the characteristics of the appendicularian unresorbed notochord, we isolated a partial genomic clone and a full-length cDNA sequence homologous to the mouse Brachyury (T) gene from the appendicularian Oikopleura longicauda. Brachyury is known to be predominantly expressed in the notochord cells and plays an important role in their differentiation in other chordates. While phylogenetic analysis robustly supports the orthology of the isolated Brachyury gene, the exon-intron organization found in the genomic clone was distinct from that well-conserved among other T-box genes. In addition to a detailed observation of notochord development in living specimens, whole-mount double in situ hybridization was carried out using a Brachyury probe along with a muscle actin probe. The Brachyury transcripts were found in the notochord of the tailbud embryos and persisted into later stages. The present study highlights characteristics of notochord development in the appendicularian. Furthermore, these results provide basic knowledge for comprehensive understanding of the cellular- and molecular-based mechanisms needed to build the characteristic cytoarchitecture of notochord that varies among tunicate species.

Structure and expression of the genes encoding proteins resident in large peripheral vesicles of Phytophthora cinnamomi zoospores.

Zoospores of Phytophthora spp. contain several characteristic types of peripheral vesicles. One of these, large peripheral vesicles, has been proposed to act as a nutrient store and in P. cinnamomi has been shown to contain three immunologically related high-molecular-weight proteins, designated LPVs. We have used antibodies directed against P. cinnamomi zoospores and cysts to isolate several cDNA clones which are products of the Lpv genes and encode one or more of the LPV proteins present in large peripheral vesicles. Northern blot analysis demonstrated the presence of three large Lpv transcripts (11-14 kb) in RNA isolated from hyphae which had been induced to form sporangia. Coordinate accumulation of the three transcripts occurred after induction of sporangium formation: no transcript was observed in uninduced hyphae and maximum transcript levels of all three transcripts were seen 4-6 h after induction. Genomic Southern blots indicated that P. cinnamomi contains three Lpv genes, presumably corresponding to the three transcripts and proteins seen in Northern and Western blots, respectively. Partial genomic clones representing two of the Lpv genes were isolated and characterized by restriction mapping and partial DNA sequencing. In the regions sequenced, the genes were > 99% identical, the high degree of conservation extending at least 415 bp downstream of their polyadenylation sites. The Lpv coding regions contained a variable number (approximately 12-18) of highly conserved 534 bp repeats, flanked by apparently unique sequences. Variation in the number of repeats in the Lpv genes was responsible for the different sizes of the three transcripts and proteins. Database searches using the Lpv nucleotide and deduced amino acid sequences failed to detect any similar sequences. We discuss the molecular events which may have been involved in the evolution of the Lpv genes and the nature of the products of these genes.

The two RNA ligases of the Trypanosoma brucei RNA editing complex: cloning the essential band IV gene and identifying the band V gene.

Kinetoplastid RNA editing is a posttranscriptional insertion and deletion of U residues in mitochondrial transcripts that involves RNA ligase. A complex of seven different polypeptides purified from Trypanosoma brucei mitochondria that catalyzes accurate RNA editing contains RNA ligases of approximately 57 kDa (band IV) and approximately 50 kDa (band V). From a partial amino acid sequence, cDNA and genomic clones of band IV were isolated, making it the first cloned component of the minimal RNA editing complex. It is indeed an RNA ligase, for when expressed in Escherichia coli, the protein autoadenylylates and catalyzes RNA joining. Overexpression studies revealed that T. brucei can regulate of total band IV protein at the level of translation or protein stability, even upon massively increased mRNA levels. The protein's mitochondrial targeting was confirmed by its location, size when expressed in T. brucei and E. coli, and N-terminal sequence. Importantly, genetic knockout studies demonstrated that the gene for band IV is essential in procyclic trypanosomes. The band IV and band V RNA ligases of the RNA editing complex therefore serve different functions. We also identified the gene for band V RNA ligase, a protein much more homologous to band IV than to other known ligases.

Enhanced expression of a calcium-dependent protein kinase from the moss Funaria hygrometrica under nutritional starvation.

Among the downstream targets of calcium in plants, calcium-dependent protein kinases (CDPKs) form an interesting class of kinases which are activated by calcium binding. They have been implicated in a diverse array of responses to hormonal and environmental stimuli. In order to dissect the role of CDPKs in the moss Funaria hygrometrica, a polymerase chain reaction (PCR)-based approach was adopted to clone the gene. Using degenerate PCR primers against conserved regions of CDPKs, a 900 bp amplicon was obtained from the genomic DNA of Funaria. Southern hybridization under low stringency conditions indicated the presence of several CDPK related sequences in the Funaria genome. This observation is consistent with reports of multigene families of CDPKs in other plants. The 900 bp fragment was subsequently used to isolate a 2.2 kb partial genomic clone of the CDPK gene from Funaria. The genomic clone encodes an open reading frame (ORF) of 518 amino acids. Interestingly, unlike other CDPK genes from plants, the entire 1.5 kb ORF is not interrupted by introns. The deduced amino acid sequence of the Funaria gene shows extensive homology with CDPKs from higher plants, 73% identity with the Fragaria CDPK and 71% identity with CDPK isoform 7 of Arabidopsis. Phylogenetic analysis revealed that the Funaria CDPK is closer to the CDPKs from higher plants like strawberry and Arabidopsis as compared to those from lower plants such as the liverwort Marchantia, the green alga Chlamydomonas or another moss Tortula. Northern analysis shows enhanced expression of the CDPK transcript within 24-48 h of starvation for nitrogen, phosphorus or sulphur. So far the only other kinase which is known to be induced by nutrient starvation in plants is the wpk 4 which is a snf-1 related kinase (SnRKs). To our knowledge this is the first report that implicates a CDPK in the starvation response.

Cloning and molecular analysis of two new sesquiterpene cyclases from Artemisia annua L.

Artemisia annua L. is the only source of artemisinin, a new promising antimalarial drug (Qinghaosu Antimalarial Coordinating Research Group, Chin. Med. J. 92 (1979) 811). Our efforts are focused on the overproduction of this valuable medicine by genetic engineered A. annua plants. Therefore, we decided to isolate the gene(s) encoding sesquiterpene cyclase(s) in A. annua as a first step in improving artemisinin yield. Four partial genomic clones, gASC21, gASC22, gASC23 and gASC24, were isolated through polymerase chain reaction (PCR) with degenerated primers based on homologous boxes present in sesquiterpene cyclases from divergent sources. Intron–exon organisation of those partial genomic clones was analysed and it was shown that A. annua contains a gene family for sesquiterpene cyclases. Based on gASC21, gASC22, gASC23 and gASC24 sequences, the full-length cDNA clones cASC34 and cASC125 were subsequently isolated by rapid amplification of cDNA ends PCR. The derived amino acid sequences of both full-length clones show high homology with sesquiterpene cyclases from plants. Reverse transcription-PCR analysis revealed transient and tissue specific expression patterns for cASC34 and cASC125, in contrast to the constitutively expressed 8-epicedrol synthase, a previously reported sesquiterpene cyclase from A. annua. Both cASC34 and cASC125 could only be detected in flowering plants when artemisinin concentration is at highest.

A simple method for generating full length cDNA from low abundance partial genomic clones

BackgroundPCR amplification of target molecules involves sequence specific primers that flank the region to be amplified. While this technique is generally routine, its applicability may not be sufficient to generate a desired target molecule from two separate regions involving intron /exon boundaries. For these situations, the generation of full-length complementary DNAs from two partial genomic clones becomes necessary for the family of low abundance genes.ResultsThe first approach we used for the isolation of full-length cDNA from two known genomic clones of Hox genes was based on fusion PCR. Here we describe a simple and efficient method of amplification for homeobox D13 (HOXD13) full length cDNA from two partial genomic clones. Specific 5' and 3' untranslated region (UTR) primer pairs and website program (primer3_www.cgv0.2) were key steps involved in this process.ConclusionsWe have devised a simple, rapid and easy method for generating cDNA clone from genomic sequences. The full length HOXD13 clone (1.1 kb) generated with this technique was confirmed by sequence analysis. This simple approach can be utilized to generate full-length cDNA clones from available partial genomic sequences.

Sequences of the waxy loci of wheat: utility in analysis of waxy proteins and developing molecular markers.

The waxy proteins from a number of genetic backgrounds of wheat and its progenitors were analyzed by SDS-PAGE. The deduced amino acid sequences of the waxy proteins of three diploid progenitor species indicated several key amino acid substitutions, which could explain the differences observed in the electrophoretic mobilities of the wheat waxy proteins. A slight difference observed in the apparent molecular weight of the WX-A1 protein of diploid and polyploid wheat was explained by amino acid substitutions or variations in predicted protein structures. Further, twelve different partial genomic clones, representing the individual waxy loci of the various diploid, tetraploid and hexaploid wheats, were isolated and compared. The results indicated significant variations in intron 4 and led to identification of sequences unique to the individual waxy genes and genomes of wheat and its proposed progenitors. The sequence variations observed have a great potential for development as molecular markers for identification of specific waxy loci and study of the various waxy mutants of wheat.

NADPH Oxidase Genes from Tomato (Lycopersicon esculentum) and Curly‐Leaf Pondweed (Potamogeton crispus)

Abstract: The oxidative burst is an integral component of plant resistance to pathogens. There is accumulating evidence that the oxidative burst is catalyzed by an enzyme with similarities to the phagocyte NADPH oxidase. We have cloned a full length homolog of the gp91 (phox) subunit of the plasma membrane NADPH oxidase complex from tomato named LeRBOM. The predicted protein contains 989 amino acids. The large N‐terminal domain contains two EF hand calcium binding motifs and one conserved glycosylation site. Six putative membrane spans are present in the C‐terminal half of the predicted protein. Extensive homology with the human gp91 (phox) subunit was found including conservation of amino acid residues important for heme coordination and substrate binding. We have also isolated partial genomic clones from tomato and from the aquatic plant Potamogeton crispus. These species serve as models for studies of signal transduction leading to NADPH oxidase activation. In tomato, LeRBOH1 expression was too low to be detected on Northern blots. RT‐PCR indicated that LeRBOH1 was expressed in all tissues tested.

Immediate Upstream Sequence of Arrestin Directs Rod-specific Expression in Xenopus

Arrestins are a family of proteins that modulate G protein-coupled receptor responses with distinct arrestin genes expressed in rods and cones. To understand the regulatory mechanisms controlling rod-specific expression, the abundant Xenopus rod arrestin cDNA and a partial genomic clone, containing the immediate upstream region and amino terminus of the polypeptide, have been characterized. The deduced polypeptide has approximately 69% identity to other vertebrate rod arrestins. Southern blot analysis and polymerase chain reaction of intronic sequences demonstrated multiple alleles for rod arrestin. DNase I footprinting with retinal proteins revealed four major DNA binding sites in the proximal promoter, coinciding with consensus sequences reported in mammalian promoters. Purified bovine Crx homeodomain and mouse Nrl proteins protected a number of these sites. A dual approach of transient embryo transfections and transgenesis was used to locate transcriptional control sequences essential for rod-specific expression in Xenopus. Constructs containing -1287/+113 of 5' upstream sequence with or without intron 1 directed high level expression, specifically in rods. A construct containing only -287/+113 directed expression of green fluorescent protein solely in rod cells. These results suggest that the Crx and Nrl binding sites in the proximal promoter are the primary cis-acting sequences regulating arrestin gene expression in rods.

Molecular investigation of aminopeptidase N expression in the winter flounder, Pleuronectes americanus

The primary structure of a polypeptide with significant similarity to human aminopeptidase N (EC 3.4.11.2) was deduced from the sequence of two overlapping cDNA clones derived from winter flounder (Pleuronectes americanus, Walbaum) intestinal RNA and a partial genomic clone derived from winter flounder DNA. The deduced amino acid sequence (975 amino acids) is comprised of an N-terminal cytosolic portion of seven amino acid residues, followed by a 24-residue transmembrane anchor region, a 38-residue serine/threonine-rich junction and a 906-residue enzymatic domain that protrudes from the apical membrane of the enterocyte. A highly conserved signature sequence characteristic of zinc-dependent metallopeptidases (HExxH) is present, as well as seven putative glycosylation sites (NxS/T). Fourteen introns are present in the 7.53 kb portion of the gene that was cloned and sequenced. Reverse transcriptase polymerase chain reaction assays using primers spanning intron/exon boundaries were used to determine the timing of expression of this gene in larval winter flounder. This represents the first aminopeptidase N sequence to be determined from a teleost fish and underscores the utility of molecular biological information in the investigation of larval fish digestion.

Otoconin-90, the mammalian otoconial matrix protein, contains two domains of homology to secretory phospholipase A2.

The ability to sense orientation relative to gravity requires dense particles, called otoconia, which are localized in the vestibular macular organs. In mammals, otoconia are composed of proteins (otoconins) and calcium carbonate crystals in a calcite lattice. Little is known about the mechanisms that regulate otoconial biosynthesis. To begin to elucidate these mechanisms, we have partially sequenced and cloned the major protein component of murine otoconia, otoconin-90 (OC90). The amino acid sequence identified an orphan chimeric human cDNA. Because of its similarity to secretory phospholipase A2 (sPLA2), this gene was referred to as PLA2-like (PLA2L) and enabled the identification of human Oc90. Partial murine cDNA and genomic clones were isolated and shown to be specifically expressed in the developing mouse otocyst. The mature mouse OC90 is composed of 453 residues and contains two domains homologous to sPLA2. The cloning of Oc90 will allow an examination of the role of this protein in otoconial biosynthesis and in diseases that affect the vestibular system.

More than 80R2R3-MYB regulatory genes in the genome of Arabidopsis thaliana.

Transcription factors belonging to the R2R3-MYB family contain the related helix-turn-helix repeats R2 and R3. The authors isolated partial cDNA and/or genomic clones of 78 R2R3-MYB genes from Arabidopsis thaliana and found accessions corresponding to 31 Arabidopsis genes of this class in databanks, seven of which were not represented in the authors' collection. Therefore, there are at least 85, and probably more than 100, R2R3-MYB genes present in the Arabidopsis thaliana genome, representing the largest regulatory gene family currently known in plants. In contrast, no more than three R2R3-MYB genes have been reported in any organism from other phyla. DNA-binding studies showed that there are differences but also frequent overlaps in binding specificity among plant R2R3-MYB proteins, in line with the distinct but often related functions that are beginning to be recognized for these proteins. This large-sized gene family may contribute to the regulatory flexibility underlying the developmental and metabolic plasticity displayed by plants.

Molecular Cloning and Functional Expression of Mannitol-1-phosphatase from the Apicomplexan Parasite Eimeria tenella

A metabolic pathway responsible for the biosynthesis and utilization of mannitol is present in the seven species of Eimeria that infect chickens, but is not in the avian host. Mannitol-1-phosphatase (M1Pase), a key enzyme for mannitol biosynthesis, is a highly substrate-specific phosphatase and, accordingly, represents an attractive chemotherapeutic target. Amino acid sequence of tryptic peptides obtained from biochemically purified Eimeria tenella M1Pase was used to synthesize degenerate oligonucleotide hybridization probes. Using these reagents, a partial genomic clone and full-length cDNA clones have been isolated and characterized. The deduced amino acid sequence of E. tenella M1Pase shows limited overall homology to members of the phosphohistidine family of phosphatases. This limited homology to other histidine phosphatases does, however, include several conserved residues that have been shown to be essential for their catalytic activity. Kinetic parameters of recombinant M1Pase expressed in bacteria are essentially identical to those of the biochemically purified preparation from E. tenella. Moreover, recombinant M1Pase is subject to active site-directed, hydroxylamine-reversible inhibition by the histidine-selective acylating reagent diethyl pyrocarbonate. These results indicate the presence of an essential histidine residue(s) at the M1Pase active site, as predicted for a histidine phosphatase.

Erythrocyte binding protein homologues of rodent malaria parasites

Erythrocyte invasion by malaria parasites requires specific molecular interactions between the merozoite and erythrocyte surface receptors. A well-conserved, functionally important family of erythrocyte binding proteins is the EBP family. The EBP family includes the Plasmodium vivax/ P. knowlesi Duffy binding protein (DBP) family and the P. falciparum erythrocyte binding antigen-175 (EBA-175). The EBP are transmembrane proteins, characterized by two conserved cysteine-rich domains, expressed in the micronemes of invasive merozoites. Oligonucleotide primers matching the region encoding the carboxyl cysteine-rich domain of the EBA-175 were used in a polymerase chain reaction to identify homologous genes in P. berghei and P. yoelii yoelii, leading to the isolation of a P. berghei partial genomic clone. This clone contained a 323 bp region that had high deduced amino acid sequence similarity to the amino acid sequences of the carboxyl cysteine-rich domains of the DBP family and EBA-175. The P. berghei carboxyl cysteine-rich domain was followed by a putative transmembrane domain and a cytoplasmic domain, demonstrating an exon–intron structure at the 3′ end homologous to P. vivax dbp and P. falciparum eba-175. The carboxyl cysteine-rich domain is also highly conserved among P. berghei, P. y. yoelii, P. chabaudi and P. vinckei and is encoded by a single copy gene. Antisera prepared against the carboxyl cysteine-rich domain of the rodent malaria EBP homologues reacted with a 120 and 128 kDa protein doublet on Western blots of P. berghei parasite antigen and showed an apical localization pattern within merozoites by indirect immunofluorescence assays.