Number Of Open Reading Frames Research Articles

Transcript profiling in Candida albicans reveals new cellular functions for the transcriptional repressors CaTup1, CaMig1 and CaNrg1.

The pathogenic fungus, Candida albicans contains homologues of the transcriptional repressors ScTup1, ScMig1 and ScNrg1 found in budding yeast. In Saccharomyces cerevisiae, ScMig1 targets the ScTup1/ScSsn6 complex to the promoters of glucose repressed genes to repress their transcription. ScNrg1 is thought to act in a similar manner at other promoters. We have examined the roles of their homologues in C. albicans by transcript profiling with an array containing 2002 genes, representing about one quarter of the predicted number of open reading frames (ORFs) in C. albicans. The data revealed that CaNrg1 and CaTup1 regulate a different set of C. albicans genes from CaMig1 and CaTup1. This is consistent with the idea that CaMig1 and CaNrg1 target the CaTup1 repressor to specific subsets of C. albicans genes. However, CaMig1 and CaNrg1 repress other C. albicans genes in a CaTup1-independent fashion. The targets of CaMig1 and CaNrg1 repression, and phenotypic analyses of nrg1/nrg1 and mig1/mig1 mutants, indicate that these factors play differential roles in the regulation of metabolism, cellular morphogenesis and stress responses. Hence, the data provide important information both about the modes of action of these transcriptional regulators and their cellular roles. The transcript profiling data are available at http://www.pasteur.fr/recherche/unites/RIF/transcriptdata/.

The complete DNA sequence of the Ectocarpus siliculosus Virus EsV-1 genome.

The Ectocarpus siliculosus Virus-1, EsV-1, is the type-species of a genus of Phycodnaviridae, the phaeoviruses, infecting marine filamentous brown algae. The EsV-1 genome of 335,593 bp contains tandem and dispersed repetitive elements in addition to a large number of open reading frames of which 231 are currently counted as genes. Many genes can be assigned to functional groups involved in DNA synthesis, DNA integration, transposition, and polysaccharide metabolism. Furthermore, EsV-1 contains components of a surprisingly complex signal transduction system with six different hybrid histidine protein kinases and four putative serine/threonine protein kinases. Several other genes encode polypeptides with protein-protein interaction domains. However, 50% of the predicted genes have no counterparts in data banks. Only 28 of the 231 identified genes have significant sequence similarities to genes of the Chlorella virus PBCV-1, another phycodnavirus. To our knowledge, the EsV-1 genome is the largest viral DNA sequenced to date.

Functional characterization of streptococcal pyrogenic exotoxin J, a novel superantigen.

Streptococcal toxic shock syndrome (STSS) is a highly lethal, acute-onset illness that is a subset of invasive streptococcal disease. The majority of clinical STSS cases have been associated with the pyrogenic toxin superantigens (PTSAgs) streptococcal pyrogenic exotoxin A or C (SPE A or C), although cases have been reported that are not associated with either of these exotoxins. Recent genome sequencing projects have revealed a number of open reading frames that potentially encode proteins with similarity to SPEs A and C and to other PTSAgs. Here, we describe the cloning, expression, purification, and functional characterization of a novel exotoxin termed streptococcal pyrogenic exotoxin J (SPE J). Purified recombinant SPE J (rSPE J) expressed from Escherichia coli stimulated the expansion of both rabbit splenocytes and human peripheral blood lymphocytes, preferentially expanded human T cells displaying Vbeta2, -3, -12, -14, and -17 on their T-cell receptors, and was active at concentrations as low as 5 x 10(-6) microg/ml. Furthermore, rSPE J induced fevers in rabbits and was lethal in two models of STSS. Biochemically, SPE J had a predicted molecular weight of 24,444 and an isoelectric point of 7.7 and lacked the ability to form the cystine loop structure characteristic of many PTSAgs. SPE J shared 19.6, 47.1, 38.8, 18.1, 19.6, and 24.4% identity with SPEs A, C, G, and H, streptococcal superantigen, and streptococcal mitogenic exotoxin Z-2, respectively, and was immunologically cross-reactive with SPE C. The characterization of a seventh functional streptococcal PTSAg raises important questions relating to the evolution of the streptococcal superantigens.

Repeat-associated phase variable genes in the complete genome sequence of Neisseria meningitidis strain MC58.

Phase variation, mediated through variation in the length of simple sequence repeats, is recognized as an important mechanism for controlling the expression of factors involved in bacterial virulence. Phase variation is associated with most of the currently recognized virulence determinants of Neisseria meningitidis. Based upon the complete genome sequence of the N. meningitidis serogroup B strain MC58, we have identified tracts of potentially unstable simple sequence repeats and their potential functional significance determined on the basis of sequence context. Of the 65 potentially phase variable genes identified, only 13 were previously recognized. Comparison with the sequences from the other two pathogenic Neisseria sequencing projects shows differences in the length of the repeats in 36 of the 65 genes identified, including 25 of those not previously known to be phase variable. Six genes that did not have differences in the length of the repeat instead had polymorphisms such that the gene would not be expected to be phase variable in at least one of the other strains. A further 12 candidates did not have homologues in either of the other two genome sequences. The large proportion of these genes that are associated with frameshifts and with differences in repeat length between the neisserial genome sequences is further corroborative evidence that they are phase variable. The number of potentially phase variable genes is substantially greater than for any other species studied to date, and would allow N. meningitidis to generate a very large repertoire of phenotypes through expression of these genes in different combinations. Novel phase variable candidates identified in the strain MC58 genome sequence include a spectrum of genes encoding glycosyltransferases, toxin related products, and metabolic activities as well as several restriction/modification and bacteriocin-related genes and a number of open reading frames (ORFs) for which the function is currently unknown. This suggests that the potential role of phase variation in mediating bacterium-host interactions is much greater than has been appreciated to date. Analysis of the distribution of homopolymeric tract lengths indicates that this species has sequence-specific mutational biases that favour the instability of sequences associated with phase variation.

Inhibition of Self-incompatible Pollen in Papaver rhoeas Involves a Complex Series of Cellular Events

Self-incompatibility (SI) in Papaver rhoeas involves allele-specific recognition between stigmatic S proteins and pollen which results in the rapid inhibition of incompatible pollen. In this article we discuss current understanding of the components and mechanisms involved in this reaction. Analysis of cDNA sequences encoding several stigmatic S alleles has allowed us to establish, using site-directed mutagenesis, that certain amino acids are essential for S-specific inhibitory activity. An S protein binding protein (SBP) identified in pollen plasma membrane extracts has been proposed to act as a receptor. Study of S1 gene mutants defective in SBP binding has confirmed the important role of SBP in the SI reaction, however the exact nature of this remains to be defined. Although it was previously thought that the Papaver stigmatic S gene was unique, detailed analysis of Arabidopsis thaliana genomic sequences has revealed the presence of a large number of open reading frames with homology to the S gene. We have named these potential genes SPH (S-protein homologues). Studies of the molecular and biochemical basis of self-incompatibility (SI) in P. rhoeas have revealed much about the early signals triggered in pollen in this response. We have established that a Ca2+-dependent signal transduction pathway mediates the inhibition of incompatible pollen. Rapid increases in cytosolic free Ca2+ and the rapid loss of the pollen apical Ca2+ gradient are accompanied by the hyperphosphorylation of p26 and p68, two soluble pollen phosphoproteins. Studies reveal that p26 is a soluble inorganic pyrophosphatase. Recent evidence has been obtained that suggests irreversible pollen tube growth and cell death involves a programmed cell death (PCD) pathway.

A Genome-Wide Screening in Saccharomyces cerevisiae for Genes That Confer Resistance to the Anticancer Agent Cisplatin

Cisplatin is a potent DNA-damaging agent that has demonstrated anticancer activities against several tumors. However, manifestation of cellular resistance is a major obstacle in anticancer therapy that severely limits the curative potential of cisplatin. Therefore, understanding the molecular basis of cisplatin resistance could significantly improve the clinical efficacy of this anticancer agent. Here, we employed Saccharomyces cerevisiae as a model organism to study cisplatin resistance mechanisms and describe a one-step cisplatin selection to identify and characterize novel cisplatin resistance genes. Screening a multicopy yeast genomic library enabled us to isolate several yeast clones for which we could confirm that the cisplatin resistance phenotype was linked to the introduced fragment. In a first attempt, a number of open reading frames could be identified. Among these genes, PDE2 and ZDS2 were repeatedly identified as genes whose overexpression confers cellular resistance to cisplatin. PDE2, encoding cAMP-phosphodiesterase 2, is of particular interest because the overexpression of this yeast gene is known to induce cisplatin resistance in mammalian cells as well, providing proof of the principle of our experimental approach. In addition, the identification of PDE2 shows that our yeast screening system can directly be informative for drug resistance in mammalian cells.

Genome-Wide Protein Interaction Screens Reveal Functional Networks Involving Sm-Like Proteins

A set of seven structurally related Sm proteins forms the core of the snRNP particles containing the spliceosomal U1, U2, U4 and U5 snRNAs. A search of the genomic sequence of Saccharomyces cerevisiae has identified a number of open reading frames that potentially encode structurally similar proteins termed Lsm (L¯ike Sm¯) proteins. With the aim of analysing all possible interactions between the Lsm proteins and any protein encoded in the yeast genome, we performed exhaustive and iterative genomic two-hybrid screens, starting with the Lsm proteins as baits. Indeed, extensive interactions amongst eight Lsm proteins were found that suggest the existence of a Lsm complex or complexes. These Lsm interactions apparently involve the conserved Sm domain that also mediates interactions between the Sm proteins. The screens also reveal functionally significant interactions with splicing factors, in particular with Prp4 and Prp24, compatible with genetic studies and with the reported association of Lsm proteins with spliceosomal U6 and U4/U6 particles. In addition, interactions with proteins involved in mRNA turnover, such as Mrt1, Dcp1, Dcp2 and Xrn1, point to roles for Lsm complexes in distinct RNA metabolic processes, that are confirmed in independent functional studies. These results provide compelling evidence that two-hybrid screens yield functionally meaningful information about protein–protein interactions and can suggest functions for uncharacterized proteins, especially when they are performed on a genome-wide scale.

34] GATEWAY recombinational cloning: Application to the cloning of large numbers of open reading frames or ORFeomes

Publisher Summary Complete genome sequences are available for three model organisms—Escherichia coli, Saccharomyces cerevisiae, and Caenorhabditis elegans—and for several pathogenic microorganisms such as Helicobacter pylori. Complete genome sequences are expected to become available soon for other model organisms and for humans. This information is expected to revolutionize the way biological questions can be addressed. Molecular mechanisms should now be approachable on a more global scale in the context of (nearly) complete sets of genes, rather than by analyzing genes individually. However, most open reading frames (ORFs) predicted from sequencing projects have remained completely uncharacterized at the functional level. The emerging field of functional genomics addresses this limitation by developing methods to characterize the function of large numbers of predicted ORFs simultaneously.

Genome-wide protein interaction screens reveal functional networks involving Sm-like proteins.

A set of seven structurally related Sm proteins forms the core of the snRNP particles containing the spliceosomal U1, U2, U4 and U5 snRNAs. A search of the genomic sequence of Saccharomyces cerevisiae has identified a number of open reading frames that potentially encode structurally similar proteins termed Lsm (Like Sm) proteins. With the aim of analysing all possible interactions between the Lsm proteins and any protein encoded in the yeast genome, we performed exhaustive and iterative genomic two-hybrid screens, starting with the Lsm proteins as baits. Indeed, extensive interactions amongst eight Lsm proteins were found that suggest the existence of a Lsm complex or complexes. These Lsm interactions apparently involve the conserved Sm domain that also mediates interactions between the Sm proteins. The screens also reveal functionally significant interactions with splicing factors, in particular with Prp4 and Prp24, compatible with genetic studies and with the reported association of Lsm proteins with spliceosomal U6 and U4/U6 particles. In addition, interactions with proteins involved in mRNA turnover, such as Mrt1, Dcp1, Dcp2 and Xrn1, point to roles for Lsm complexes in distinct RNA metabolic processes, that are confirmed in independent functional studies. These results provide compelling evidence that two-hybrid screens yield functionally meaningful information about protein–protein interactions and can suggest functions for uncharacterized proteins, especially when they are performed on a genome-wide scale.

Microbial genomics

The 1.6-Mb sequence of the genome of the aerobic hyperthermophilic crenarchaeon Aeropyrum pernix K1 has been published by staff at the National Institute of Technology and Evaluation, Tokyo, Japan 1 Kawarabayasi, Y. et al. (1999) Complete genome sequence of an aerobic hyper-thermophilic crenarchaeon, Aeropyrum pernix K1, DNA Res. 6, 83–101, 145–152 Google Scholar . A. pernix was first isolated from a coastal solfataric thermal vent on Kodakara-Jima Island, Japan in 1993, grows optimally at 95°C and was the first strictly aerobic hyperthermophilic archaeon to be described. Of the 2694 open reading frames (ORFs) identified in the genome sequence, over half do not show any significant similarity to known sequences. All the enzymes of the tricarboxylic-acid cycle are encoded in the genome, except for α-ketoglutarate dehydrogenase (which might be functionally replaced by 2-oxoacid:ferredoxin oxidoreductase). Intragenomic sequence comparisons suggest that a considerable number of ORFs were generated by sequence duplication. Fourteen of the 50 RNA genes contain introns. An online genome database for A. pernix is available on http://www.mild.nite.go.jp/. The genome sequence is also available in seven segments from GenBank (http://www.ncbi.nlm.nih.gov/Genbank/index.html)

Transcriptional analysis of the hmw gene cluster of Mycoplasma pneumoniae.

Mycoplasma pneumoniae adherence to host cells is a multifactorial process that requires the cytadhesin P1 and additional accessory proteins. The hmw gene cluster consists of the genes p30, hmw3, and hmw1, the products of which are known to be essential for cytadherence, the rpsD gene, and six open reading frames of unknown function. Putative transcriptional terminators flank this locus, raising the possibility that these genes are expressed as a single transcriptional unit. However, S1 nuclease protection and primer extension experiments identified probable transcriptional start sites upstream of the p32, p21, p50, and rpsD genes. Each was preceded at the appropriate spacing by the -10-like sequence TTAAAATT, but the -35 regions were not conserved. Analysis of the M. pneumoniae genome sequence indicated that this promoter-like sequence is found upstream of only a limited number of open reading frames, including the genes for P65 and P200, which are structurally related to HMW1 and HMW3. Promoter deletion studies demonstrated that the promoter-like region upstream of p21 was necessary for the expression of p30 and an hmw3-cat fusion in M. pneumoniae, while deletion of the promoter-like region upstream of p32 had no apparent effect. Analysis by reverse transcription-PCR confirmed transcriptional linkage of all the open reading frames in the hmw gene cluster. Taken together, these findings suggest that the genes of this locus constitute an operon expressed from overlapping transcripts.

The DmpA aminopeptidase from Ochrobactrum anthropi LMG7991 is the prototype of a new terminal nucleophile hydrolase family

The DmpA (d-aminopeptidase A) protein produced by Ochrobactrum anthropi hydrolyses p-nitroanilide derivatives of glycine and d-alanine more efficiently than that of l-alanine. When regular peptides are utilized as substrates, the enzyme behaves as an aminopeptidase with a preference for N-terminal residues in an l configuration, thus exemplifying an interesting case of stereospecificity reversal. The best-hydrolysed substrate is l-Ala-Gly-Gly, but tetra- and penta-peptides are also efficiently hydrolysed. The gene encodes a 375-residue precursor, but the active enzyme contains two polypeptides corresponding to residues 2-249 (alpha-subunit) and 250-375 (beta-subunit) of the precursor. Residues 249 and 250 are a Gly and a Ser respectively, and various substitutions performed by site-directed mutagenesis result in the production of an uncleaved and inactive protein. The N-terminal Ser residue of the beta-subunit is followed by a hydrophobic peptide, which is predicted to form a beta-strand structure. All these properties strongly suggest that DmpA is an N-terminal amidohydrolase. An exploration of the databases highlights the presence of a number of open reading frames encoding related proteins in various bacterial genomes. Thus DmpA is very probably the prototype of an original family of N-terminal hydrolases.

The DmpA aminopeptidase from Ochrobactrum anthropi LMG7991 is the prototype of a new terminal nucleophile hydrolase family

The DmpA (D-aminopeptidase A) protein produced by Ochrobactrum anthropi hydrolyses p-nitroanilide derivatives of glycine and D-alanine more efficiently than that of L-alanine. When regular peptides are utilized as substrates, the enzyme behaves as an aminopeptidase with a preference for N-terminal residues in an L configuration, thus exemplifying an interesting case of stereospecificity reversal. The best-hydrolysed substrate is L-Ala-Gly-Gly, but tetra- and penta-peptides are also efficiently hydrolysed. The gene encodes a 375-residue precursor, but the active enzyme contains two polypeptides corresponding to residues 2-249 (α-subunit) and 250-375 (β-subunit) of the precursor. Residues 249 and 250 are a Gly and a Ser respectively, and various substitutions performed by site-directed mutagenesis result in the production of an uncleaved and inactive protein. The N-terminal Ser residue of the β-subunit is followed by a hydrophobic peptide, which is predicted to form a β-strand structure. All these properties strongly suggest that DmpA is an N-terminal amidohydrolase. An exploration of the databases highlights the presence of a number of open reading frames encoding related proteins in various bacterial genomes. Thus DmpA is very probably the prototype of an original family of N-terminal hydrolases.

Analysis of Arabidopsis genome sequence reveals a large new gene family in plants.

A detailed analysis of the currently available Arabidopsis thaliana genomic sequence has revealed the presence of a large number of open reading frames with homology to the stigmatic self-incompatibility (S) genes of Papaver rhoeas. The products of these potential genes are all predicted to be relatively small, basic, secreted proteins with similar predicted secondary structures. We have named these potential genes SPH (S-protein homologues). Their presence appears to have been largely missed by the prediction methods currently used on the genomic sequence. Equivalent homologues could not be detected in the human, microbial, Drosophila or C. elegans genomic databases, suggesting a function specific to plants. Preliminary RT-PCR analysis indicates that at least two members of the family (SPH1, SPH8) are expressed, with expression being greatest in floral tissues. The gene family may total more than 100 members, and its discovery not only illustrates the importance of the genome sequencing efforts, but also indicates the extent of information which remains hidden after the initial trawl for potential genes.

High-level expression of soluble heterologous proteins in the cytoplasm of Escherichia coli by fusion to the bacteriophage lambda head protein D.

The bacteriophage Lambda head protein D (gpD) is a small major capsid protein (110aa; 11.6kDa; pI=5.68, devoid of cysteine residues) that is essential for stable head morphogenesis. We found that a His-tagged derivative of gpD (gpHD) is a monomeric protein with efficient expression properties and high resistance towards thermally induced irreversible aggregation. In addition, gpHD can be used as a fusion partner for high-level expression of soluble heterologous proteins in the cytoplasm of Escherichia coli. Its broad utility is illustrated by the production of various mammalian proteins by fusion to its C-terminus. As a fusion partner, gpHD is thought to mediate optimal translation initiation while reducing inclusion body formation and protein degradation. In addition, it provides a His-tag for simple purification. gpHD may act as a 'cytoplasmic anchor' by keeping its unfolded fusion partner in solution, thereby providing more time for proper folding. An ever-increasing number of open reading frames (ORFs) are being identified in the various genome sequencing programs. gpHD has the potential to be harnessed for the development of highly efficient cytoplasmic expression systems that might contribute to the production and characterization of these novel polypeptides. Protein D is also an established fusion partner for phage display. It thus presents the attractive opportunity of coupling the selection of heterologous proteins from a phage library to their subsequent high-level expression.

Complete sequence and gene organization of the genome of a hyper-thermophilic archaebacterium, Pyrococcus horikoshii OT3 (supplement).

The complete sequence of the genome of a hyper-thermophilic archaebacterium, Pyrococcus horikoshii OT3, has been determined by assembling the sequences of the physical map-based contigs of fosmid clones and of long polymerase chain reaction (PCR) products which were used for gap-filling. The entire length of the genome was 1,738,505 bp. The authenticity of the entire genome sequence was supported by restriction analysis of long PCR products, which were directly amplified from the genomic DNA. As the potential protein-coding regions, a total of 2061 open reading frames (ORFs) were assigned, and by similarity search against public databases, 406 (19.7%) were related to genes with putative function and 453 (22.0%) to the sequences registered but with unknown function. The remaining 1202 ORFs (58.3%) did not show any significant similarity to the sequences in the databases. Sequence comparison among the assigned ORFs in the genome provided evidence that a considerable number of ORFs were generated by sequence duplication. By similarity search, 11 ORFs were assumed to contain the intein elements. The RNA genes identified were a single 16S-23S rRNA operon, two 5S rRNA genes and 46 tRNA genes including two with the intron structure. All the assigned ORFs and RNA coding regions occupied 91.25% of the whole genome. The data presented in this paper are available on the internet at http://www.nite.go.jp.

Immunological and biochemical characterization of membrane proteins.

AbstractMembrane proteins of mycoplasmas assume particular significance in investigations of the pathogenesis of disease. They are involved in adhesion to, and probably in other interactions with, the cells of the target tissues of the host (1–5). They are frequently significant antigens in the immune response (4–7), and in some cases have been observed to undergo high-frequency size and/or phase variation (8–13). In addition, many membrane proteins are also important in metabolic pathways, including transmembrane transport (14). The significance of future research on membrane proteins of mycoplasmas has been recently highlighted by the completion of the genomic sequences of Mycoplasma genitalium and Mycoplasma pneumoniae (15,16). In both species, the products of a number of open reading frames could not be assigned a function by comparison to known prokaryotic genes. The vast majority of these unassigned products were putative membrane proteins.KeywordsPowdered Skim MilkLuria BrothIsoamyl AlcoholHydrophobic FractionCircular PieceThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Complete sequence and gene organization of the genome of a hyper-thermophilic archaebacterium, Pyrococcus horikoshii OT3.

The complete sequence of the genome of a hyper-thermophilic archaebacterium, Pyrococcus horikoshii OT3, has been determined by assembling the sequences of the physical map-based contigs of fosmid clones and of long polymerase chain reaction (PCR) products which were used for gap-filling. The entire length of the genome was 1,738,505 bp. The authenticity of the entire genome sequence was supported by restriction analysis of long PCR products, which were directly amplified from the genomic DNA. As the potential protein-coding regions, a total of 2061 open reading frames (ORFs) were assigned, and by similarity search against public databases, 406 (19.7%) were related to genes with putative function and 453 (22.0%) to the sequences registered but with unknown function. The remaining 1202 ORFs (58.3%) did not show any significant similarity to the sequences in the databases. Sequence comparison among the assigned ORFs in the genome provided evidence that a considerable number of ORFs were generated by sequence duplication. By similarity search, 11 ORFs were assumed to contain the intein elements. The RNA genes identified were a single 16S-23S rRNA operon, two 5S rRNA genes and 46 tRNA genes including two with the intron structure. All the assigned ORFs and RNA coding regions occupied 91.25% of the whole genome. The data presented in this paper are available on the internet at http:@www.nite.go.jp.

Novel Vibrio cholerae O139 genes involved in lipopolysaccharide biosynthesis.

The sequence of part of the rfb region of Vibrio cholerae serogroup O139 and the physical map of a 35-kb region of the O139 chromosome have been determined. The O139 rfb region presented contains a number of open reading frames which show similarities to other rfb and capsular biosynthesis genes found in members of the Enterobacteriaceae family and in V. cholerae O1. The cloned and sequenced region can complement the defects in O139 antigen biosynthesis in transposon insertions within the O139 rfb cluster. Linkage is demonstrated among IS1358 of V. cholerae O139, the rfb region, and the recently reported otnA and otnB genes (E. M. Bik, A. E. Bunschoten, R. D. Gouw, and F. R. Mooi, EMBO J. 14:209-216, 1995). In addition, the whole of this region has been linked to the rfaD gene. Furthermore, determination of the sequence flanking IS1358 has revealed homology to other rfb-like genes. The exact site of insertion with respect to rfaD is defined for the novel DNAs of both the Bengal and the Argentinian O139 isolates.

M-T2: a poxvirus TNF receptor homologue with dual activities.

Poxviruses are experts at manipulating and evading the host's immune response. They have acquired a number of open reading frames which specifically confer direct anti-immune properties, either by mimicking cytokine receptors and growth factors or by disarming cytokine regulatory cascades. The Myxoma T2 protein (M-T2), a TNF receptor homologue, is secreted from virus infected cells and can bind TNF-alpha with high affinity, and thereby inhibit TNF-alpha-mediated cytotoxicity. M-T2 also acts to inhibit virus-induced lymphocyte apoptosis by an as yet undefined mechanism. As such, T2 constitutes a significant virulence factor for poxviruses, influencing the outcome of infection, both in vitro and in vivo.