Medicinal Plants: Guests and Hosts in the Heterologous Expression of High-Value Products.

Medicinal plants play an important dual role in the context of the heterologous expression of high-value pharmaceutical products. On the one hand, the classical biochemical and modern omics approaches allowed for the discovery of various genes encoding biosynthetic pathways in medicinal plants. Recombinant DNA technology enabled introducing these genes and regulatory elements into host organisms and enhancing the heterologous production of the corresponding secondary metabolites. On the other hand, the transient expression of foreign DNA in plants facilitated the production of numerous proteins of pharmaceutical importance. This review summarizes several success stories of the engineering of plant metabolic pathways in heterologous hosts. Likewise, a few examples of recombinant protein expression in plants for therapeutic purposes are also highlighted. Therefore, the importance of medicinal plants has grown immensely as sources for valuable products of low and high molecular weight. The next step ahead for bioengineering is to achieve more success stories of industrial-scale production of secondary plant metabolites in microbial systems and to fully exploit plant cell factories' commercial potential for recombinant proteins.

Assessing the fate of recombinant plant DNA in rabbit's tissues fed genetically modified cotton.

Various feeding studies have been conducted with the different species of animals to evaluate the possible transfer of transgenic DNA (tDNA) from genetically modified (GM) feed into the animal tissues. However, the conclusions drawn from most of such studies are sometimes controversial. Thus, in the present study, an attempt has been made to evaluate the fate of tDNA in rabbits raised on GM cotton-based diet through PCR analysis of the DNA extracted specifically from blood, liver, kidney, heart and intestine (jejunum). A total of 48 rabbits were fed a mixed diet consisting variable proportions of transgenic cottonseeds meal (i.e. 0% w/w, 20% w/w, 30% w/w and 40% w/w) for 180 days. The presence of transgenic DNA fragments (Cry1Ac, Cry2A and CP4 EPSPS) or plant endogenous gene (Sad1) was traced in those specific tissues and organs. The presence of ß-actin (ACTB) was also monitored as an internal control. Neither the transgenic fragments (459 bp of Cry1Ac gene, 167 bp of Cry2A gene and111 bp of CP4 EPSPS gene) nor cotton endogenous reference gene (155 bp of Sad1) could be detected in any of the DNA samples extracted from the rabbit's tissues in both control and transgenic groups. However, 155 bp fragment of the rabbit's reference gene (ACTB) was recovered in all the DNA samples extracted from rabbit tissues. The results obtained from this study revealed that both plant endogenous and transgenic DNA fragments have same fate in rabbit's tissues and were efficiently degraded in the gastrointestinal tract (GIT).

Establishment of Cre-mediated HBV recombinant cccDNA (rcccDNA) cell line for cccDNA biology and antiviral screening assays.

Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA), existing in hepatocyte nuclei as a stable minichromosome, plays a central role in the life cycle of the virus and permits the persistence of infection. Despite being essential for HBV infection, little is known about the molecular mechanisms of cccDNA formation, regulation and degradation, and there is no therapeutic agents directly targeting cccDNA, fore mostly due to the lack of robust, reliable and quantifiable HBV cccDNA models. In this study, combined the Cre/loxP and sleeping beauty transposons system, we established HepG2-derived cell lines integrated with 2-60 copies of monomeric HBV genome flanked by loxP sites (HepG2-HBV/loxP). After Cre expression via adenoviral transduction, 3.3-kb recombinant cccDNA (rcccDNA) bearing a chimeric intron can be produced in the nuclei of these HepG2-HBV/loxP cells. The rcccDNA could be accurately quantified by quantitative PCR using specific primers and cccDNA pool generated in this model could be easily detected by Southern blotting using the digoxigenin probe system. We demonstrated that the rcccDNA was epigenetically organized as the natural minichromosome and served as the template supporting pgRNA transcription and viral replication. As the expression of HBV S antigen (HBsAg) is dependent on the newly generated cccDNA, HBsAg is the surrogate marker of cccDNA. Additionally, the efficacies of 3 classes of anti-HBV agents were evaluated in HepG2-HBV/loxP cells and antiviral activities with different mechanisms were confirmed. These data collectively suggested that HepG2-HBV/loxP cell system will be powerful platform for studying cccDNA related biological mechanisms and developing novel cccDNA targeting drugs.

Structural Insights into the HIV-1 Minus-strand Strong-stop DNA.

An essential step of human immunodeficiency virus type 1 (HIV-1) reverse transcription is the first strand transfer that requires base pairing of the R region at the 3'-end of the genomic RNA with the complementary r region at the 3'-end of minus-strand strong-stop DNA (ssDNA). HIV-1 nucleocapsid protein (NC) facilitates this annealing process. Determination of the ssDNA structure is needed to understand the molecular basis of NC-mediated genomic RNA-ssDNA annealing. For this purpose, we investigated ssDNA using structural probes (nucleases and potassium permanganate). This study is the first to determine the secondary structure of the full-length HIV-1 ssDNA in the absence or presence of NC. The probing data and phylogenetic analysis support the folding of ssDNA into three stem-loop structures and the presence of four high-affinity binding sites for NC. Our results support a model for the NC-mediated annealing process in which the preferential binding of NC to four sites triggers unfolding of the three-dimensional structure of ssDNA, thus facilitating interaction of the r sequence of ssDNA with the R sequence of the genomic RNA. In addition, using gel retardation assays and ssDNA mutants, we show that the NC-mediated annealing process does not rely on a single pathway (zipper intermediate or kissing complex).

Construction of chimera protein by using artificial restriction DNA cutter.

We have already developed artificial restriction DNA cutter (ARCUT), which can hydrolyze double-stranded DNA site-selectively, by using Ce(IV)/EDTA in combination with two pseudo-complementary peptide nucleic acids (pcPNAs). Here, ARCUT was used to prepare a chimera protein. The gene for WW-domain containing oxidoreductase (WWOX) was clipped off by ARCUT just before its stop codon, and ligated with the gene for enhanced green fluorescent protein (EGFP). Conventional PCR for insertion of restriction enzyme site is never required.

Spread of recombinant DNA by roots and pollen of transgenic potato plants, identified by highly specific biomonitoring using natural transformation of an Acinetobacter sp.

Transgenic potato plants with the nptII gene coding for neomycin phosphotransferase (kanamycin resistance) as a selection marker were examined for the spread of recombinant DNA into the environment. We used the recombinant fusion of nptII with the tg4 terminator for a novel biomonitoring technique. This depended on natural transformation of Acinetobacter sp. strain BD413 cells having in their genomes a terminally truncated nptII gene (nptII'; kanamycin sensitivity) followed by the tg4 terminator. Integration of the recombinant fusion DNA by homologous recombination in nptII' and tg4 restored nptII, leading to kanamycin-resistant transformants. DNA of the transgenic potato was detectable with high sensitivity, while no transformants were obtained with the DNA of other transgenic plants harboring nptII in different genetic contexts. The recombinant DNA was frequently found in rhizosphere extracts of transgenic potato plants from field plots. In a series of field plot and greenhouse experiments we identified two sources of this DNA: spread by roots during plant growth and by pollen during flowering. Both sources also contributed to the spread of the transgene into the rhizospheres of nontransgenic plants in the vicinity. The longest persistence of transforming DNA in field soil was observed with soil from a potato field in 1997 sampled in the following year in April and then stored moist at 4 degrees C in the dark for 4 years prior to extract preparation and transformation. In this study natural transformation is used as a reliable laboratory technique to detect recombinant DNA but is not used for monitoring horizontal gene transfer in the environment.

Antisense suppression of a potato alpha-SNAP homologue leads to alterations in cellular development and assimilate distribution.

Using the cDNA-AFLP method, we have isolated a transcript-derived fragment (TDF) which shows a differential expression pattern during tuber organogenesis of Solanum tuberosum L. The TDF was used to isolate a cDNA clone carrying a 1.5 kb insert and potentially coding for a 32.5 kDa peptide which, by homology, represents a potato homologue of an alpha-snap gene and has been designated Stsnap. Northern analysis showed that the Stsnap gene is expressed in actively dividing tissues throughout the potato plant. Analysis of genomic DNA from potato revealed that the Stsnap gene is likely to be a single-copy gene. The expression of antisense Stsnap cDNA under the control of the CaMV 35S promoter results in plants with an altered morphology such as curled leaves. Several of these transgenic lines also display cellular and developmental abnormalities with distinct changes in assimilate transport including accumulation of starch and soluble sugars in source leaves. We argue that these findings are consistent with the hypothetical function of the StSNAP gene product in vesicle targeting and fusion during plant development.

Molecular cloning of a ferret angiotensin II AT(1) receptor reveals the importance of position 163 for Losartan binding.

A complementary DNA for the angiotensin II (AngII) type 1 (AT(1)) receptor from Mustela putorius furo (ferret) was isolated from a ferret atria cDNA library. The cDNA encodes a protein (fAT(1)) of 359 amino acids having high homologies (93-99%) to other mammalian AT(1) receptor counterparts. When fAT(1) was expressed in COS-7 cells and photoaffinity labeled with the photoactive analogue (125)I-¿Sar(1), Bpa(8)AngII, a protein of 100 kDa was detected by autoradiography. The formation of this complex was specific since it was abolished in the presence of the AT(1) non-peptidic antagonist L-158,809. Functional analysis indicated that the fAT(1) receptor efficiently coupled to phospholipase C as demonstrated by an increase in inositol phosphate production following stimulation with AngII. Binding studies revealed that the fAT(1) receptor had a high affinity for the peptide antagonist ¿Sar(1), Ile(8)AngII (K(d) of 5. 8+/-1.4 nM) but a low affinity for the AT(1) selective non-peptidic antagonist DuP 753 (K(d) of 91+/-15.6 nM). Interestingly, when we substituted Thr(163) with an Ala residue, which occupies this position in many mammalian AT(1) receptors, we restored the high affinity of this receptor for Dup 753 (11.7+/-5.13 nM). These results suggest that position 163 of the AT(1) receptor does not contribute to the overall binding of peptidic ligands but that certain non-peptidic antagonists such as Dup 753 are clearly dependent on this position for efficient binding.

Matrix metalloproteinase MMP-19 (RASI-1) is expressed on the surface of activated peripheral blood mononuclear cells and is detected as an autoantigen in rheumatoid arthritis.

In order to characterize the autoimmune response participating in the pathogenesis of rheumatoid arthritis (RA) a cDNA expression library constructed from mRNAs which had been isolated from the inflamed synovium of an RA patients was screened with autologous IgG autoantibodies. This led to the identification of gene rasi-1 which encodes a protein showing sequence identity with the zinc-binding matrix metalloproteinase MMP-19. MMP-19 is detected on the surface of activated PBMCs, TH1 lymphocytes, and Jurkat T lymphoma cells. It exhibits gelatinolytic activity and is recognized by autoantibodies in 26% and, respectively, 33% of sera collected from RA patients and systemic lupus erythematosus (SLE) patients. The novel autoantigen MMP-19 thus could play a role in the pathological processes participating in RA-associated joint tissue destruction.

The binding of UvrAB proteins to bubble and loop regions in duplex DNA.

Based on the binding of the UvrAB complex to a promoter region in transcription open complexes (Ahn, B., and Grossman, L. (1996) J. Biol. Chem. 271, 21453-21461) and the requirement of a single-stranded region for UvrAB helicase activity, we examined the binding of UvrAB proteins to synthetic bubble or loop regions in duplex DNA and the role of these regions in translocation of the UvrAB complex as well as incision of DNA damage. We found that the UvrAB complex was able to bind to bubble and loop regions with an affinity similar to that for damaged DNA in the absence of RNAP. The preferential recognition and incision of damaged sites by the UvrAB complex was observed downstream of the bubble or loop region in the strand complementary to the strand along which the UvrAB complex translocates. These results imply that the bubble region generated in duplex DNA by RNAP serves as a preferred entry site for the translocation of the UvrAB complex, and that preferential binding and unidirectional translocation of the UvrAB complex predetermine where incision is to occur.

The MHC class II molecule H2-M is targeted to an endosomal compartment by a tyrosine-based targeting motif.

The nonpolymorphic human class II molecule HLA-DM (DM) has been found to play a key role in antigen presentation by MHC class II molecules. HLA-DM and its murine equivalent H2-M are located intracellularly and are absent from the cell surface. In transfected HeLa cells, H2-M was transported to an endosomal compartment in the absence of invariant chain. A tyrosine-based targeting motif in the cytoplasmic tail of H2-M beta was responsible for the endosomal location and, if this tyrosine was mutated, H2-M accumulated at the cell surface. In the presence of invariant chain the mutated H2-M was redistributed to endosomes. The targeting motif of H2-M appeared not to be crucial for efficient peptide loading of class II, but if the invariant chain targeting motif also was removed, peptide loading decreased drastically. Thus, the targeting motif of H2-M appears to be supplementary, rather than essential for class II-peptide association.

Features of the formate dehydrogenase mRNA necessary for decoding of the UGA codon as selenocysteine.

The fdhF gene encoding the 80-kDa selenopolypeptide subunit of formate dehydrogenase H from Escherichia coli contains an in-frame TGA codon at amino acid position 140, which encodes selenocysteine. We have analyzed how this UGA "sense codon" is discriminated from a UGA codon signaling polypeptide chain termination. Deletions were introduced from the 3' side into the fdhF gene and the truncated 5' segments were fused in-frame to the lacZ reporter gene. Efficient read-through of the UGA codon, as measured by beta-galactosidase activity and incorporation of selenium, was dependent on the presence of at least 40 bases of fdhF mRNA downstream of the UGA codon. There was excellent correlation between the results of the deletion studies and the existence of a putative stem-loop structure lying immediately downstream of the UGA in that deletions extending into the helix drastically reduced UGA translation. Similar secondary structures can be formed in the mRNAs coding for other selenoproteins. Selenocysteine insertion cartridges were synthesized that contained this hairpin structure and variable portions of the fdhF gene upstream of the UGA codon and inserted into the lacZ gene. Expression studies showed that upstream sequences were not required for selenocysteine insertion but that they may be involved in modulating the efficiency of read-through. Translation of the UGA codon was found to occur with high fidelity since it was refractory to ribosomal mutations affecting proofreading and to suppression by the sup-9 gene product.

Steroid hormone regulation of ribosomal RNA in rat hypothalamus: early detection using in situ hybridization and precursor-product ribosomal DNA probes.

In the female rat, behavioral and endocrine aspects of reproduction are controlled, in part, by the action of the steroid hormone estradiol on several regions of the brain, including the ventrolateral portion of the ventromedial hypothalamus (VL-VMN) and the arcuate nucleus of the hypothalamus (ARC). Quantitative assessment of the effects of estradiol on the regulation of ribosomal RNA in rat hypothalamus was accomplished in this study by tandem in situ hybridization experiments with 2 ribosomal DNA probes specific to the initial transcript (precursor) or mature, stable (product) rRNA. This novel approach allowed the regulation of RNA processing by steroid hormones to be analyzed in the individual neuron, a particularly important concern in heterogeneous tissue such as the brain. Estradiol was administered subcutaneously to ovariectomized rats for 15 min, 30 min, or 2 hr, or a discontinuous schedule of 2 hr on/7 hr off/2 hr on. Levels of precursor and product rRNA were measured in VL-VMN and ARC neurons using a computerized image-analysis system. Significant increases in the levels of precursor rRNA were observed only in the VL-VMN as early as 30 min after hormone exposure, with a doubling in the amount of precursor rRNA occurring at 2 hr. No changes in product rRNA were observed in either brain region at these early times. These data, in conjunction with our previous findings of increases in product rRNA after longer hormone exposure times, lead us to conclude that rRNA gene transcription is activated in rat hypothalamic neurons within 30 min.(ABSTRACT TRUNCATED AT 250 WORDS)

Chloroplast and cytosolic glutamine synthetase are encoded by homologous nuclear genes which are differentially expressed in vivo.

We have shown that the individual members of the plant gene family for glutamine synthetase (GS) are differentially expressed in vivo, and each encode distinct GS polypeptides which are targeted to different subcellular compartments (chloroplast or cytosol). At the polypeptide level, chloroplast GS (GS2) and cytosolic GS (GS1 and GSn) are distinct and show an organ-specific distribution. We have characterized full length cDNA clones encoding chloroplast or cytosolic GS of pea. In vitro translation products encoded by three different GS cDNA clones, correspond to the mature GS2, GS1, and GSn polypeptides present in vivo. pGS185 encodes a precursor to the chloroplast GS2 polypeptide as shown by in vitro chloroplast uptake experiments. The pGS185 translation product is imported into the chloroplast stroma and processed to a polypeptide which corresponds in size and charge to that of mature chloroplast stromal GS2 (44 kDa). The 49 amino terminal amino acids encoded by pGS185 are designated as a chloroplast transit peptide by functionality in vitro, and amino acid homology to other transit peptides. The cytosolic forms of GS (GS1 and GSn) are encoded by highly homologous but distinct mRNAs. pGS299 encodes the cytosolic GS1 polypeptide (38 kDa), while pGS341 (Tingey, S. V., Walker, E. L., and Coruzzi, G. M. (1987) EMBO. J. 6, 1-9) encodes a cytosolic GSn polypeptide (37 kDa). The homologous nuclear genes for chloroplast and cytosolic GS show different patterns of expression in vivo. GS2 expression in leaves is modulated by light, at the level of steady state mRNA and protein, while the expression of cytosolic GS is unaffected by light. The light-induced expression of GS2 is due at least in part to a phytochrome mediated response. Nucleotide sequence analysis indicates that chloroplast and cytosolic GS have evolved from a common ancestor and suggest a molecular mechanism for chloroplast evolution.

Construction and analysis of DNA sequence libraries from flow-sorted chromosomes: practical and theoretical considerations.

We describe the construction and analysis of recombinant DNA libraries representative of chromosomes 1 and 2 of Chinese hamster (Cricetulus griseus). Propidium-iodide stained chromosomes were purified by flow cytometric analysis and sorting, and EcoRI digests of purified DNA were cloned into the bacteriophage vector Charon 4A. These libraries contain DNA complementary to 63% and 69% of nick-translated DNA derived from flow-purified chromosomes 1 and 2, respectively. However, sequences complementary to only 24% and 35% of a total Chinese hamster genomic DNA tracer were hybridized in parallel renaturation experiments. The chromosome 2 library contained DNA sequences encoding dihydrofolate reductase (dhfr), a gene previously mapped to Chinese hamster chromosome 2. No sequences complementary to dhfr were found in the library constructed from chromosome 1 DNA. These analyses are discussed with regard to the current limitations and future strategies for the construction of chromosome-specific DNA sequence libraries of high purity and completeness.

An in vitro method generating base substitutions in preselected regions of plasmid DNA: application to structural analysis of the replication origin of the Escherichia coli K-12 chromosome.

A method for introducing base substitutions in defined regions of plasmid DNA has been developed. In principle, a circular heteroduplex DNA containing a gap is constructed by annealing of two kinds of linear molecules derived from the same plasmid: One is the molecule shortened either by exonucleolytic digestion from the termini generated at a restriction site or by removal of a region flanked by two restriction sites, and the other the full-length molecule linearized at a different site. The deleted region in the shorter linear molecule becomes a single-stranded gap in the circular heteroduplex DNA. The heteroduplex is then treated with sodium bisulfite that converts specifically cytosine residues to uracil residues in single-stranded regions. After filling in the gap by repair synthesis, transformation is carried out to isolate mutant plasmids. Since two kinds of circular heteroduplexes are formed by annealing in which the sequences in the gaps are complementary to each other, mutagenesis of both strands can be accomplished in one experiment. This method was applied to construction of mutants with base substitutions in the replication origin region (oriC) of the Escherichia coli K-12 chromosome which had previously been cloned in colicin E1 plasmid vectors, and various mutants in defined regions of oriC were successfully isolated at high efficiencies. Analysis of these mutants provided evidence that oriC contains special regions, designated spacers, which separate neighboring important sequences specifying interactions with initiation factors for DNA replication at precise distances.

Calcitonin messenger RNA encodes multiple polypeptides in a single precursor.

Recombinant DNA techniques were used to analyze the structure of the messenger RNA encoding a precursor of calcitonin, a small calcium-regulating hormone of 32 amino acids. Analyses of the nucleotide sequences of cloned complementary DNA's comprising the entire coding sequence of the messenger RNA revealed that calcitonin is flanked at both its amino and carboxyl termini by peptide extensions linked to the hormone by short sequences of basic amino acids. The location of glycine next to the carboxyl terminal prolinamide of calcitonin is consistent with indications that glycine is required for the enzymatic amidation of proline to the prolinamide. During cellular biosynthesis, calcitonin arises from a large precursor protein by cleavages at both amino and carboxyl terminal residues of the hormone. These findings raise questions concerning the regulation of these cleavages and the potential biological functions of the precursor extensions derived from these cleavages.

Gene amplification causes overproduction of the first three enzymes of UMP synthesis in N-(phosphonacetyl)-L-aspartate-resistant hamster cells.

Mutant Syrian hamster cells resistant to N-(phosphonacetyl)-L-aspartate (PALA), a transition state analog inhibitor of aspartate transcarbamylase, overproduce CAD, a multifunctional protein which catalyzes the first three reactions of de novo UMP biosynthesis. Increased levels of a single mRNA cause the overproduction of CAD in all PALA-resistant mutants examined thus far. A recombinant plasmid containing a 2,3-kilobase insert complementary to the 3'-proximal region of this 7.9-kilobase mRNA has been prepared and used to show that the CAD gene is amplified in each of the 10 PALA-resistant mutants examined. Rates of association of CAD sequences in DNA isolated from PALA-sensitive and PALA-resistant cells with labeled plasmid DNA indicated that the degree of amplification is approximately equal to the degree of overproduction of protein and mRNA in each mutant. The patterns of digestion of these DNAs with restriction enzymes confirmed this result and showed that the lower limit for the size of the amplified unit is 19 kilobases, much larger than the mRNA. A comparison of restriction endonuclease digests of the cloned cDNA with digests of genomic DNA indicated that part of this difference is attributable to intervening sequences in the CAD gene. A 10.2-kilobase RNA which contains CAD sequences is found in cytoplasmic fractions from some PALA-resistant mutants but not in wild type cells. Restriction patterns were analyzed by a new method in which fragments of DNA are transferred from agarose gels to diazo paper with a high efficiency which is independent of size.