Targeted gene mutation in tetraploid potato through transient TALEN expression in protoplasts.

Potato is the third largest food crop in the world, however, the high degree of heterozygosity, the tetrasomic inheritance and severe inbreeding depression are major difficulties for conventional potato breeding. The rapid development of modern breeding methods offers new possibilities to enhance breeding efficiency and precise improvement of desirable traits. New site-directed mutagenesis techniques that can directly edit the target genes without any integration of recombinant DNA are especially favorable. Here we present a successful pipeline for site-directed mutagenesis in tetraploid potato through transient TALEN expression in protoplasts. The transfection efficiency of protoplasts was 38-39% and the site-directed mutation frequency was 7-8% with a few base deletions as the predominant type of mutation. Among the protoplast-derived calli, 11-13% showed mutations and a similar frequency (10%) was observed in the regenerated shoots. Our results indicate that the site-directed mutagenesis technology could be used as a new breeding method in potato as well as for functional analysis of important genes to promote sustainable potato production.

Antibacterial mechanism of fraxetin against Staphylococcus aureus.

Fraxetin is one of the main constituents of the traditional medicinal plant Fraxinus rhynchophylla. The inhibitory effect of fraxetin on various bacterial strains has been extensively reported, however, its mechanism of action on bacterial cells remains to be elucidated. In the present study, the antibacterial mechanism of fraxetin on Staphylococcus aureus was systematically investigated by examining its effect on cell membranes, protein synthesis, nucleic acid content and topoisomerase activity. The results indicated that fraxetin increased the permeability of the cell membrane but did not render it permeable to macromolecules, such as DNA and RNA. Additionally, the quantity of protein, DNA and RNA decreased to 55.74, 33.86 and 48.96%, respectively following treatment with fraxetin for 16 h. The activity of topoisomerase I and topoisomerase II were also markedly inhibited as fraxetin concentration increased. The result of the ultraviolet­visible spectrophotometry demonstrated that the DNA characteristics exhibited a blue shift and hypochromic effect following treatment with fraxetin. These results indicated that fraxetin had a marked inhibitory effect on S.aureus proliferation. Further mechanistic studies showed that fraxetin could disrupt nucleic acid and protein synthesis by preventing topoisomerase from binding to DNA.

[Restriction endonuclease digest - melting curve analysis: a new SNP genotyping and its application in traditional Chinese medicine authentication].

Single nucleotide polymorphisms (SNP) is an important molecular marker in traditional Chinese medicine research, and it is widely used in TCM authentication. The present study created a new genotyping method by combining restriction endonuclease digesting with melting curve analysis, which is a stable, rapid and easy doing SNP genotyping method. The new method analyzed SNP genotyping of two chloroplast SNP which was located in or out of the endonuclease recognition site, the results showed that when attaching a 14 bp GC-clamp (cggcgggagggcgg) to 5' end of the primer and selecting suited endonuclease to digest the amplification products, the melting curve of Lonicera japonica and Atractylodes macrocephala were all of double peaks and the adulterants Shan-yin-hua and A. lancea were of single peaks. The results indicated that the method had good stability and reproducibility for identifying authentic medicines from its adulterants. It is a potential SNP genotyping method and named restriction endonuclease digest - melting curve analysis.

Development of an enzyme-linked-receptor assay based on Syrian hamster ß2-adrenergic receptor for detection of ß-agonists.

ß-Adrenergic agonists (ß-agonists) are illegally used in animal husbandry, threatening the health of consumers. To realize multianalyte detection of ß-agonists, a ß2-adrenergic receptor (ß2-AR) was cloned from Syrian hamster lung and heterogeneously expressed by Spodoptera frugiperda (Sf9) cells. The recombinant ß2-AR was purified from intracellular soluble proteins of infected Sf9 cells, and was utilized to establish an enzyme-linked-receptor assay (ELRA) to detect a group of ß-agonists simultaneously. This assay was based on direct competitive inhibition of binding of horseradish peroxidase-labeled ractopamine to the immobilized ß2-AR proteins by ß-agonists. The IC50 and limit of detection values for ractopamine were 30.38µgL(-1) and 5.20µgL(-1), respectively. Clenbuterol and salbutamol showed 87.7% and 58.5% cross-reactivities with ractopamine, respectively. This assay is simple, rapid, and environmentally friendly, showing a potential application in the screening of ß-agonists in animal feeds.

Site-selective scission of human genome using PNA-based artificial restriction DNA cutter.

Site-selective scission of genomes is quite important for future biotechnology. However, naturally occurring restriction enzymes cut these huge DNAs at too many sites and cannot be used for this purpose. Recently, we have developed a completely chemistry-based artificial restriction DNA cutter (ARCUT) by combining a pair of pseudo-complementary PNA (pcPNA) strands (sequence recognition moiety) and Ce(IV)/EDTA complex (molecular scissors). The scission site of ARCUT and its scission specificity can be freely modulated in terms of the sequences and lengths of the pcPNA strands so that even huge genomes can be selectively cut at only one predetermined site. In this chapter, the method of site-selective scission of human genomic DNA using ARCUT is described in detail.

Contrasting arbuscular mycorrhizal communities colonizing different host plants show a similar response to a soil phosphorus concentration gradient.

High soil phosphorus (P) concentration is frequently shown to reduce root colonization by arbuscular mycorrhizal (AM) fungi, but the influence of P on the diversity of colonizing AM fungi is uncertain. We used terminal restriction fragment length polymorphism (T-RFLP) of 18S rDNA and cloning to assess diversity of AM fungi colonizing maize (Zea mays), soybean (Glycene max) and field violet (Viola arvensis) at three time points in one season along a P gradient of 10-280 mg l(-1) in the field. Percentage AM colonization changed between sampling time points but was not reduced by high soil P except in maize. There was no significant difference in AM diversity between sampling time points. Diversity was reduced at concentrations of P > 25 mg l(-1), particularly in maize and soybean. Both cloning and T-RFLP indicated differences between AM communities in the different host species. Host species was more important than soil P in determining the AM community, except at the highest P concentration. Our results show that the impact of soil P on the diversity of AM fungi colonizing plants was broadly similar, despite the fact that different plants contained different communities. However, subtle differences in the response of the AM community in each host were evident.

Green tea polyphenols function as prooxidants to inhibit Pseudomonas aeruginosa and induce the expression of oxidative stress-related genes.

Green tea polyphenols (GTP) are widely believed to function as antioxidants and antimicrobial agents. Here we observed that GTP and epigallocatechin gallate, the most abundant catechin in GTP, could also function as prooxidants and produce hydrogen peroxide (H2O2) to inhibit the growth of Pseudomonas aeruginosa. pH value of the medium was the key factor that affected prooxidant versus antioxidant property of GTP. Under weakly acidic conditions (pH 5.5-6.5), GTP showed antioxidant activity by eliminating H2O2; whereas, under neutral and weakly alkaline conditions (pH 7.0-8.0), GTP showed prooxidant activity and inhibited the growth of P. aeruginosa. Furthermore, we studied the effects of GTP on gene expression profiles of a few oxidative stress-related genes by quantitative real-time PCR analysis. After 10 min to 1 h of exposure under weakly alkaline condition, GTP significantly up-regulated expression levels of katB, sodM, ohr, lexA, and recN gene. These findings highlight that the pH-dependent H2O2 production by GTP contributes to the antibacterial activity and can induce oxidative stress-related responses in P. aeruginosa.

The Type ISP Restriction-Modification enzymes LlaBIII and LlaGI use a translocation-collision mechanism to cleave non-specific DNA distant from their recognition sites.

The Type ISP Restriction-Modification (RM) enzyme LlaBIII is encoded on plasmid pJW566 and can protect Lactococcus lactis strains against bacteriophage infections in milk fermentations. It is a single polypeptide RM enzyme comprising Mrr endonuclease, DNA helicase, adenine methyltransferase and target-recognition domains. LlaBIII shares >95% amino acid sequence homology across its first three protein domains with the Type ISP enzyme LlaGI. Here, we determine the recognition sequence of LlaBIII (5'-TnAGCC-3', where the adenine complementary to the underlined base is methylated), and characterize its enzyme activities. LlaBIII shares key enzymatic features with LlaGI; namely, adenosine triphosphate-dependent DNA translocation (∼309 bp/s at 25°C) and a requirement for DNA cleavage of two recognition sites in an inverted head-to-head repeat. However, LlaBIII requires K(+) ions to prevent non-specific DNA cleavage, conditions which affect the translocation and cleavage properties of LlaGI. By identifying the locations of the non-specific dsDNA breaks introduced by LlaGI or LlaBIII under different buffer conditions, we validate that the Type ISP RM enzymes use a common translocation-collision mechanism to trigger endonuclease activity. In their favoured in vitro buffer, both LlaGI and LlaBIII produce a normal distribution of random cleavage loci centred midway between the sites. In contrast, LlaGI in K(+) ions produces a far more distributive cleavage profile.

Using ITS2 PCR-RFLP to generate molecular markers for authentication of Sophora flavescens Ait.

BACKGROUND: Dried root of Sophora flavescens Ait. is a medicinal material occasionally misused or adulterated by other species similar in appearance. In this study the internal transcribed spacer (ITS) regions of DNA samples of S. flavescens Ait. collected from different areas of Taiwan were amplified by polymerase chain reaction (PCR) and compared. The effectiveness of using ITS2 PCR restriction fragment length polymorphism (RFLP)-generated markers to differentiate S. flavescens Ait. from possible adulterants was also evaluated. RESULTS: The S. flavescens Ait. samples collected from different areas were extremely low in ITS sequence variability at species level. ITS2 PCR-RFLP coupled with restriction enzymes Sac I, Sac II, Xho I or Pvu I produced specific fragments for all tested variants. ITS2 PCR-RFLP coupled with Sac II was further performed to identify mixtures of DNA extracts of S. flavescens Ait. and Sophora tomentosa L. in various ratios. The developed ITS2 PCR-RFLP markers could detect mixed DNA samples of S. flavescens Ait./S. tomentosa L. up to a ratio of 10:1. CONCLUSION: The present study demonstrates the usefulness of ITS2 PCR-RFLP coupled with pre-selected restriction enzymes for practical and accurate authentication of S. flavescens Ait. The technique is also suitable for analysing S. flavescens Ait. mixed with other adulterants.

Creation and validation of a ligation-independent cloning (LIC) retroviral vector for stable gene transduction in mammalian cells.

BACKGROUND: Cloning vectors capable of retroviral transduction have enabled stable gene overexpression in numerous mitotic cell lines. However, the relatively small number of feasible restriction enzyme sequences in their cloning sites can hinder successful generation of overexpression constructs if these sequences are also present in the target cDNA insert. RESULTS: Utilizing ligation-independent cloning (LIC) technology, we have modified the highly efficient retroviral transduction vector, pBABE, to eliminate reliance on restriction enzymes for cloning. Instead, the modified plasmid, pBLIC, utilizes random 12/13-base overhangs generated by T4 DNA polymerase 3' exonuclease activity. PCR-based introduction of the complementary sequence into any cDNA of interest enables universal cloning into pBLIC. Here we describe creation of the pBLIC plasmid, and demonstrate successful cloning and protein overexpression from three different cDNAs, Bax, catalase, and p53 through transduction into the human prostate cancer cell line, LNCaP or the human lung cancer line, H358. CONCLUSIONS: Our results show that pBLIC vector retains the high transduction efficiency of the original pBABE while eliminating the requirement for checking individual cDNA inserts for internal restriction sites. Thus it comprises an effective retroviral cloning system for laboratory-scale stable gene overexpression or for high-throughput applications such as creation of retroviral cDNA libraries. To our knowledge, pBLIC is the first LIC vector for retroviral transduction-mediated stable gene expression in mammalian cells.

Photo-activated DNA binding and antimicrobial activities of alkaloids from Glycosmis pentaphylla.

In our screening for photosensitizers from natural resources, four alkaloids were isolated from Glycosmis pentaphylla by various chromatography techniques. Their structures were identified as glycoborinine (1), glybomine B (2), carbalexin A (3) and N-p-coumaroyltyramine (4) by spectral analysis. Their photoactivated antimicrobial activities were evaluated by thin-layer chromatography (TLC) agar overlay assay against Staphylococcus aureus and Bacillus subtilis. It was found that compounds 1 and 4 showed photo-activated antimicrobial activities. Meantime, photo-activated DNA binding activities of these compounds were also assessed by using a specially prepared 1.8 kb DNA fragment and restriction enzymes. Under UVA irradiation, compound 1 showed moderate inhibition on Nde I, Xba I, Nco I and Bcl I which have either 5'-TpA or 5'-ApT and trace or no inhibition on other restriction enzymes. It showed a similar inhibition pattern with the reference 8-methoxypsoralen. However, compounds 2-4 showed no inhibition against any of the restriction enzymes.

Enzymatic synthesis of long double-stranded DNA labeled with haloderivatives of nucleobases in a precisely pre-determined sequence.

BACKGROUND: Restriction endonucleases are widely applied in recombinant DNA technology. Among them, enzymes of class IIS, which cleave DNA beyond recognition sites, are especially useful. We use BsaI enzyme for the pinpoint introduction of halogen nucleobases into DNA. This has been done for the purpose of anticancer radio- and phototherapy that is our long-term objective. RESULTS: An enzymatic method for synthesizing long double-stranded DNA labeled with the halogen derivatives of nucleobases (Hal-NBs) with 1-bp accuracy has been put forward and successfully tested on three different DNA fragments containing the 5-bromouracil (5-BrU) residue. The protocol assumes enzymatic cleavage of two Polymerase-Chain-Reaction (PCR) fragments containing two recognition sequences for the same or different class IIS restriction endonucleases, where each PCR fragment has a partially complementary cleavage site. These sites are introduced using synthetic DNA primers or are naturally present in the sequence used. The cleavage sites are not compatible, and therefore not susceptible to ligation until they are partially filled with a Hal-NB or original nucleobase, resulting in complementary cohesive end formation. Ligation of these fragments ultimately leads to the required Hal-NB-labeled DNA duplex. With this approach, a synthetic, extremely long DNA fragment can be obtained by means of a multiple assembly reaction (n × maximum PCR product length: n × app. 50 kb). CONCLUSIONS: The long, precisely labeled DNA duplexes obtained behave in very much the same manner as natural DNA and are beyond the range of chemical synthesis. Moreover, the conditions of synthesis closely resemble the natural ones, and all the artifacts accompanying the chemical synthesis of DNA are thus eliminated. The approach proposed seems to be completely general and could be used to label DNA at multiple pre-determined sites and with halogen derivatives of any nucleobase. Access to DNAs labeled with Hal-NBs at specific position is an indispensable condition for the understanding and optimization of DNA photo- and radio-degradation, which are prerequisites for clinical trials of Hal-NBs in anticancer therapy.

Use of primer selection and restriction enzymes to assess bacterial community diversity in an agricultural soil used for potato production via terminal restriction fragment length polymorphism.

Terminal restriction fragment length polymorphism (T-RFLP) can be used to assess how land use management changes the dominant members of bacterial communities. We compared T-RFLP profiles obtained via amplification with forward primers (27, 63F) each coupled with the fluorescently labeled reverse primer (1392R) and multiple restriction enzymes to determine the best combination for interrogating soil bacterial populations in an agricultural soil used for potato production. Both primer pairs provide nearly universal recognition of a 1,400-bp sequence of the bacterial domain in the V(1)-V(3) region of the 16S ribosomal RNA (rRNA) gene relative to known sequences. Labeling the reverse primer allowed for direct comparison of each forward primer and the terminal restriction fragments' relative migration units obtained with each primer pair and restriction enzyme. Redundancy analysis (RDA) and nested multivariate analysis of variance (MANOVA) were used to assess the effects of primer pair and choice of restriction enzyme on the measured relative migration units. Our research indicates that the 63F-1392R amplimer pair provides a more complete description with respect to the bacterial communities present in this potato (Solanum tuberosum L.)-barley (Hordeum vulgare L.) rotation over seeded to crimson clover (Trifolium praense L.). Domain-specific 16S rRNA gene primers are rigorously tested to determine their ability to amplify across a target region of the gene. Yet, variability within or between T-RFLP profiles can result from factors independent of the primer pair. Therefore, researchers should use RDA and MANOVA analyses to evaluate the effects that additional laboratory and environmental variables have on bacterial diversity.

Phosphorus-nitrogen compounds. 21. Syntheses, structural investigations, biological activities, and DNA interactions of new N/O spirocyclic phosphazene derivatives. The NMR behaviors of chiral phosphazenes with stereogenic centers upon the addition of chiral solvating agents.

The reactions of hexachlorocyclotriphosphazatriene, N(3)P(3)Cl(6), with N/O-donor-type N-alkyl (or aryl)-o-hydroxybenzylamines (1a-1e) produce mono- (2a-2e), di- (3a-3d), and tri- (4a and 4b) spirocyclic phosphazenes. The tetrapyrrolidino monospirocyclic phosphazenes (2f-2i) are prepared from the reactions of partly substituted compounds (2a-2d) with excess pyrrolidine. The dispirodipyrrolidinophosphazenes (3e-3h) and trispirophosphazenes (3i-3k) are obtained from the reactions of trans-dispirophosphazenes with excess pyrrolidine and sodium (3-amino-1-propanoxide), respectively. Compounds 3a-3d have cis and trans geometric isomers. Only the trans isomers of these compounds are isolated. Compounds 3a-3h have two stereogenic P atoms. They are expected to be in cis (meso) and trans (racemic) geometric isomers. In the trans trispiro compounds (3i-3k), there are three stereogenic P atoms. They are expected to be in racemic mixtures. The stereogenic properties of 3a-3k are confirmed by (31)P NMR spectroscopy upon the addition of the chiral solvating agent; (S)-(+)-2,2,2-trifluoro-1-(9'-anthryl)ethanol. The molecular structures of 3i-3k, 4a, and 4b look similar to a propeller, where the chemical environment of one P atom is different from that of others. Additionally, 4a and 4b are also expected to exist as cis-trans-trans and cis-cis-cis geometric isomers, but both of them are found to be in cis-trans-trans geometries. The solid-state structures of 2a, 2e, 2f, 3e, and 3f are determined by X-ray crystallography. The compounds 2f-2i, 3e-3i, and 3k are screened for antibacterial activity against gram-positive and gram-negative bacteria and for antifungal activity against yeast strains. These compounds (except 3f) have shown a strong affinity against most of the bacteria. Minimum inhibitory concentrations (MIC) are determined for 2f-2i, 3e-3i, and 3k. DNA binding and the nature of interaction with pUC18 plasmid DNA are studied. The compounds 2f-2i, 3e-3i, and 3k induce changes on the DNA mobility. The prevention of BamHI and HindIII digestion (except 2g) with compounds indicates that the compounds bind with nucleotides in DNA.

Genetic variants in the vitamin d receptor are associated with advanced prostate cancer at diagnosis: findings from the prostate testing for cancer and treatment study and a systematic review.

Low levels of plasma vitamin D have been implicated as a possible risk factor for both prostate cancer incidence and advanced disease, and recent phase II trials suggest that vitamin D supplementation might delay progression of prostate cancer. Common polymorphisms in the vitamin D receptor (VDR) are associated with VDR activity and are therefore potentially useful proxies for assessing whether vitamin D is causally related to advanced prostate cancer. We genotyped five well-known VDR polymorphisms in 1,604 men with prostate cancer from the Prostate Testing for Cancer and Treatment study. Our aim was to examine the association between VDR polymorphisms and cancer stage (localized versus advanced) as well as cancer grade (Gleason score <7 versus >or=7). Moreover, we also carried out a systematic review and meta-analysis of 13 similar studies. As a result of our meta-analysis, we revealed three polymorphisms, BsmI, ApaI, and TaqI, associated with high Gleason score with an overall summary odds ratios (95% confidence intervals) of 1.12 (1.00-1.25; bb versus BB + Bb), 1.25 (1.02-1.53; aa versus AA + Aa), and 0.82 (0.69-0.98; Tt + tt versus TT), respectively. The haplotype analysis revealed that the BsmI (B)-ApaI (A)-TaqI (t) participants compared with BsmI (b)-ApaI (a)-TaqI (T) individuals were less likely to have high Gleason scores (odds ratio, 0.84; 95% confidence interval, 0.71-1.00; P(unadjusted) = 0.050; P(adjusted) = 0.014). Our finding provides some support for the hypothesis that low levels of vitamin D may increase the risk of prostate cancer progression.

Adaptation and impairment of DNA repair function in pollen of Betula verrucosa and seeds of Oenothera biennis from differently radionuclide-contaminated sites of Chernobyl.

BACKGROUND AND AIMS: The plants that have remained in the contaminated areas around Chernobyl since 1986 encapsulate the effects of radiation. Such plants are chronically exposed to radionuclides that they have accumulated internally as well as to alpha-, beta- and gamma-emitting radionuclides from external sources and from the soil. This radiation leads to genetic damage that can be countered by DNA repair systems. The objective of this study is to follow DNA repair and adaptation in haploid cells (birch pollen) and diploid cells (seed embryos of the evening primrose) from plants that have been growing in situ in different radionuclide fall-out sites in monitored regions surrounding the Chernobyl explosion of 1986. METHODS: Radionuclide levels in soil were detected using gamma-spectroscopy and radiochemistry. DNA repair assays included measurement of unscheduled DNA synthesis, electrophoretic determination of single-strand DNA breaks and image analysis of rDNA repeats after repair intervals. Nucleosome levels were established using an ELISA kit. KEY RESULTS: Birch pollen collected in 1987 failed to perform unscheduled DNA synthesis, but pollen at gamma/beta-emitter sites has now recovered this ability. At a site with high levels of combined alpha- and gamma/beta-emitters, pollen still exhibits hidden damage, as shown by reduced unscheduled DNA synthesis and failure to repair lesions in rDNA repeats properly. Evening primrose seed embryos generated on plants at the same gamma/beta-emitter sites now show an improved DNA repair capacity and ability to germinate under abiotic stresses (salinity and accelerated ageing). Again those from combined alpha- and gamma/beta-contaminated site do not show this improvement. CONCLUSIONS: Chronic irradiation at gamma/beta-emitter sites has provided opportunities for plant cells (both pollen and embryo cells) to adapt to ionizing irradiation and other environmental stresses. This may be explained by facilitation of DNA repair function.

One-base excess adaptor ligation method for walking uncloned genomic DNA.

This report describes a novel and efficient method for walking the sequence of a genomic deoxyribonucleic acid (DNA) from a known region to an unknown region based on an oligodeoxynucleotide (oligo) cassette-mediated polymerase chain reaction technique. In this method, genomic DNA is digested by a restriction enzyme that generates a sticky 5'-end, followed by ligation of a one-base excess oligo-adaptor using T4 DNA ligase. The adaptor consists of two complementary oligos that form the same sticky end as the digested genomic DNA fragments, except that the 5'-overhang base overlaps the corresponding 3'-end base of the restriction site. This overhanging terminal base prevents ligation between the adaptors, and the appropriate molar ratio of adaptor to genomic DNA enables specific amplification of the target sequence. T4 DNA ligase catalyzes both the ligation of the phosphorylated overhang base of the adaptor to genomic DNA and the excision of the corresponding 3'-terminal base of the genomic DNA. This sequence-specific exonuclease activity of T4 DNA ligase was confirmed by ligation of an alternative adaptor in which the 5'-terminal base was not consistent with the corresponding 3'-terminal base. Using this technique, the 3'- and 5'-flanking sequences of the catalase gene of the ciliate Paramecium bursaria were determined.

Highly active artificial restriction enzyme composed of Ce(IV)/EDTA and PNA bearing phosphate group--relationship between the promotion by phosphate and the structure of invasion complex.

Recently, we developed artificial restriction DNA cutter (ARCUT) composed of pseudo-complementary peptide nucleic acid (pcPNA) and Ce(IV)/EDTA complex (EDTA = ethylenediamine-N,N,N',N'-tetraacetate). Here we promoted the site-selective hydrolysis by attaching phosphate groups to the pcPNAs. The promotion by the phosphates increased with decreasing length of the gap-like site. Furthermore, the scission was successful even when phosphate groups were introduced to 0 base-gap system.

Characterization of transport mechanisms and determinants critical for Na+-dependent Pi symport of the PiT family paralogs human PiT1 and PiT2.

The general phosphate need in mammalian cells is accommodated by members of the P(i) transport (PiT) family (SLC20), which use either Na(+) or H(+) to mediate inorganic phosphate (P(i)) symport. The mammalian PiT paralogs PiT1 and PiT2 are Na(+)-dependent P(i) (NaP(i)) transporters and are exploited by a group of retroviruses for cell entry. Human PiT1 and PiT2 were characterized by expression in Xenopus laevis oocytes with (32)P(i) as a traceable P(i) source. For PiT1, the Michaelis-Menten constant for P(i) was determined as 322.5 +/- 124.5 microM. PiT2 was analyzed for the first time and showed positive cooperativity in P(i) uptake with a half-maximal activity constant for P(i) of 163.5 +/- 39.8 microM. PiT1- and PiT2-mediated Na(+)-dependent P(i) uptake functions were not significantly affected by acidic and alkaline pH and displayed similar Na(+) dependency patterns. However, only PiT2 was capable of Na(+)-independent P(i) transport at acidic pH. Study of the impact of divalent cations Ca(2+) and Mg(2+) revealed that Ca(2+) was important, but not critical, for NaP(i) transport function of PiT proteins. To gain insight into the NaP(i) cotransport function, we analyzed PiT2 and a PiT2 P(i) transport knockout mutant using (22)Na(+) as a traceable Na(+) source. Na(+) was transported by PiT2 even without P(i) in the uptake medium and also when P(i) transport function was knocked out. This is the first time decoupling of P(i) from Na(+) transport has been demonstrated for a PiT family member. Moreover, the results imply that putative transmembrane amino acids E(55) and E(575) are responsible for linking P(i) import to Na(+) transport in PiT2.

Discovery of a novel restriction endonuclease by genome comparison and application of a wheat-germ-based cell-free translation assay: PabI (5'-GTA/C) from the hyperthermophilic archaeon Pyrococcus abyssi.

To search for restriction endonucleases, we used a novel plant-based cell-free translation procedure that bypasses the toxicity of these enzymes. To identify candidate genes, the related genomes of the hyperthermophilic archaea Pyrococcus abyssi and Pyrococcus horikoshii were compared. In line with the selfish mobile gene hypothesis for restriction-modification systems, apparent genome rearrangement around putative restriction genes served as a selecting criterion. Several candidate restriction genes were identified and then amplified in such a way that they were removed from their own translation signal. During their cloning into a plasmid, the genes became connected with a plant translation signal. After in vitro transcription by T7 RNA polymerase, the mRNAs were separated from the template DNA and translated in a wheat-germ-based cell-free protein synthesis system. The resulting solution could be directly assayed for restriction activity. We identified two deoxyribonucleases. The novel enzyme was denoted as PabI, purified and found to recognize 5'-GTAC and leave a 3'-TA overhang (5'-GTA/C), a novel restriction enzyme-generated terminus. PabI is active up to 90 degrees C and optimally active at a pH of around 6 and in NaCl concentrations ranging from 100 to 200 mM. We predict that it has a novel 3D structure.