A simple, rapid, high-fidelity and cost-effective PCR-based two-step DNA synthesis method for long gene sequences

Chemical synthesis of DNA sequences provides a powerful tool for modifying genes and for studying gene function, structure and expression. Here, we report a simple, high-fidelity and cost-effective PCR-based two-step DNA synthesis (PTDS) method for synthesis of long segments of DNA. The method involves two steps. (i) Synthesis of individual fragments of the DNA of interest: ten to twelve 60mer oligonucleotides with 20 bp overlap are mixed and a PCR reaction is carried out with high-fidelity DNA polymerase Pfu to produce DNA fragments that are ~500 bp in length. (ii) Synthesis of the entire sequence of the DNA of interest: five to ten PCR products from the first step are combined and used as the template for a second PCR reaction using high-fidelity DNA polymerase pyrobest, with the two outermost oligonucleotides as primers. Compared with the previously published methods, the PTDS method is rapid (5–7 days) and suitable for synthesizing long segments of DNA (5–6 kb) with high G + C contents, repetitive sequences or complex secondary structures. Thus, the PTDS method provides an alternative tool for synthesizing and assembling long genes with complex structures. Using the newly developed PTDS method, we have successfully obtained several genes of interest with sizes ranging from 1.0 to 5.4 kb.     

Identification of a pocket in the PDK1 kinase domain that interacts with PIF and the C-terminal residues of PKA

The 3-phosphoinositide-dependent protein kinase-1 (PDK1) phosphorylates and activates a number of protein kinases of the AGC subfamily. The kinase domain of PDK1 interacts with a region of protein kinase C-related kinase-2 (PRK2), termed the PDK1-interacting fragment (PIF), through a hydrophobic motif. Here we identify a hydrophobic pocket in the small lobe of the PDK1 kinase domain, separate from the ATP- and substrate-binding sites, that interacts with PIF. Mutation of residues predicted to form part of this hydrophobic pocket either abolished or significantly diminished the affinity of PDK1 for PIF. PIF increased the rate at which PDK1 phosphorylated a synthetic dodecapeptide (T308tide), corresponding to the sequences surrounding the PDK1 phosphorylation site of PKB. This peptide is a poor substrate for PDK1, but a peptide comprising T308tide fused to the PDK1-binding motif of PIF was a vastly superior substrate for PDK1. Our results suggest that the PIF-binding pocket on the kinase domain of PDK1 acts as a 'docking site', enabling it to interact with and enhance the phosphorylation of its substrates.     

Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies

16S ribosomal RNA gene (rDNA) amplicon analysis remains the standard approach for the cultivation-independent investigation of microbial diversity. The accuracy of these analyses depends strongly on the choice of primers. The overall coverage and phylum spectrum of 175 primers and 512 primer pairs were evaluated in silico with respect to the SILVA 16S/18S rDNA non-redundant reference dataset (SSURef 108 NR). Based on this evaluation a selection of ‘best available’ primer pairs for Bacteria and Archaea for three amplicon size classes (100–400, 400–1000, ≥1000 bp) is provided. The most promising bacterial primer pair (S-D-Bact-0341-b-S-17/S-D-Bact-0785-a-A-21), with an amplicon size of 464 bp, was experimentally evaluated by comparing the taxonomic distribution of the 16S rDNA amplicons with 16S rDNA fragments from directly sequenced metagenomes. The results of this study may be used as a guideline for selecting primer pairs with the best overall coverage and phylum spectrum for specific applications, therefore reducing the bias in PCR-based microbial diversity studies.     

Mechanism of phosphorylation of protein kinase B/Akt by a constitutively active 3-phosphoinositide-dependent protein kinase-1

Phosphorylation of Thr(308) in the activation loop and Ser(473) at the carboxyl terminus is essential for protein kinase B (PKB/Akt) activation. However, the biochemical mechanism of the phosphorylation remains to be characterized. Here we show that expression of a constitutively active mutant of mouse 3-phosphoinositide-dependent protein kinase-1 (PDK1(A280V)) in Chinese hamster ovary cells overexpressing the insulin receptor was sufficient to induce PKB phosphorylation at Thr(308) to approximately the same extent as insulin stimulation. Phosphorylation of PKB by PDK1(A280V) was not affected by treatment of cells with inhibitors of phosphatidylinositol 3-kinase or by deletion of the pleckstrin homology (PH) domain of PKB. C(2)-ceramide, a cell-permeable, indirect inhibitor of PKB phosphorylation, did not inhibit PDK1(A280V)-catalyzed PKB phosphorylation in cells and had no effect on PDK1 activity in vitro. On the other hand, co-expression of full-length protein kinase C-related kinase-1 (PRK1/PKN) or 2 (PRK2) inhibited PDK1(A280V)-mediated PKB phosphorylation. Replacing alanine at position 280 with valine or deletion of the PH domain enhanced PDK1 autophosphorylation in vitro. However, deletion of the PH domain of PDK1(A280V) significantly reduced PDK1(A280V)-mediated phosphorylation of PKB in cells. In resting cells, PDK1(A280V) localized in the cytosol and at the plasma membrane. However, PDK1(A280V) lacking the PH domain localized predominantly in the cytosol. Taken together, our findings suggest that the wild-type PDK1 may not be constitutively active in cells. In addition, activation of PDK1 is sufficient to phosphorylate PKB at Thr(308) in the cytosol. Furthermore, the PH domain of PDK1 may play both positive and negative roles in regulating the in vivo function of the enzyme. Finally, unlike the carboxyl-terminal fragment of PRK2, which has been shown to bind PDK1 and allow the enzyme to phosphorylate PKB at both Thr(308) and Ser(473), full-length PRK2 and its related kinase PRK1/PKN may both play negative roles in PKB-mediated downstream biological events.     

Identification of a PKB/Akt hydrophobic motif Ser-473 kinase as DNA-dependent protein kinase

Full activation of protein kinase B (PKB)/Akt requires phosphorylation on Thr-308 and Ser-473 by 3-phosphoinositide-dependent kinase-1 (PDK1) and Ser-473 kinase (S473K), respectively. Although PDK1 has been well characterized, the identification of the S473K remains controversial. A major PKB Ser-473 kinase activity was purified from the membrane fraction of HEK293 cells and found to be DNA-dependent protein kinase (DNA-PK). DNA-PK co-localized and associated with PKB at the plasma membrane. In vitro, DNA-PK phosphorylated PKB on Ser-473, resulting in a approximately 10-fold enhancement of PKB activity. Knockdown of DNA-PK by small interfering RNA inhibited Ser-473 phosphorylation induced by insulin and pervanadate. DNA-PK-deficient glioblastoma cells did not respond to insulin at the level of Ser-473 phosphorylation; this effect was restored by complementation with the human PRKDC gene. We conclude that DNA-PK is a long sought after kinase responsible for the Ser-473 phosphorylation step in the activation of PKB.     

3-磷酸肌醇依赖性蛋白激酶-1激活AGC家族激酶的机制研究进展

3磷酸肌醇依赖性蛋白激酶1(3phosphoinositidedependentproteinkinase1,PDK1PDPK1)是蛋白激酶B(proteinkinaseB,PKBCAKT)的上游激酶,通过与3,4,5三磷酸磷脂酰肌醇[PtdIns(3,4,5)P3]作用激活相邻的PKB分子.同时,PDK1被称为AGC激酶的掌管者(master),能够激活包含PKB在内的一系列的AGC激酶家族成员.PDK1磷酸化这些激酶的保守区域Tloop区,使它们充分激活,从而调节细胞代谢,生长,扩散,生存,抗凋亡等诸多生理过程.本文就PDK1调节AGC激酶的活性,与功能上命名的PDK2的关系,PDK1分子自身的调节,PH结构域对自身活性及AGC激酶活性的影响,PDK1定位以及作为一个新药物靶标等方面做了综述.     

In vivo role of the PIF-binding docking site of PDK1 defined by knock-in mutation

PKB/Akt, S6K, SGK and RSK are mediators of responses triggered by insulin and growth factors and are activated following phosphorylation by 3-phosphoinositide-dependent protein kinase-1 (PDK1). To investigate the importance of a substrate-docking site in the kinase domain of PDK1 termed the 'PIF-pocket', we generated embryonic stem (ES) cells in which both copies of the PDK1 gene were altered by knock-in mutation to express a form of PDK1 retaining catalytic activity, in which the PIF-pocket site was disrupted. The knock-in ES cells were viable, mutant PDK1 was expressed at normal levels and insulin-like growth factor 1 induced normal activation of PKB and phosphorylation of the PKB substrates GSK3 and FKHR. In contrast, S6K, RSK and SGK were not activated, nor were physiological substrates of S6K and RSK phosphorylated. These experiments establish the importance of the PIF-pocket in governing the activation of S6K, RSK, SGK, but not PKB, in vivo. They also illustrate the power of knock-in technology to probe the physiological roles of docking interactions in regulating the specificity of signal transduction pathways.     

The Croonian Lecture 1998. Identification of a protein kinase cascade of major importance in insulin signal transduction

Diabetes affects 3% of the European population and 140 million people worldwide, and is largely a disease of insulin resistance in which the tissues fail to respond to this hormone. This emphasizes the importance of understanding how insulin signals to the cell's interior. We have recently dissected a protein kinase cascade that is triggered by the formation of the insulin 'second messenger' phosphatidylinositide (3,4,5) trisphosphate (PtdIns (3,4,5)P3) and which appears to mediate many of the metabolic actions of this hormone. The first enzyme in the cascade is termed 3-phosphoinositide-dependent protein kinase-1 (PDK1), because it only activates protein kinase B (PKB), the next enzyme in the pathway, in the presence of PtdIns (3,4,5)P3. PKB then inactivates glycogen synthase kinase-3 (GSK3). PDK1, PKB and GSK3 regulate many physiological events by phosphorylating a variety of intracellular proteins. In addition, PKB plays an important role in mediating protection against apoptosis by survival factors, such as insulin-like growth factor-1.     

Dissecting the role of the 3-phosphoinositide-dependent protein kinase-1 (PDK1) signalling pathways

The 3-phosphoinositide-dependent protein kinase-1 (PDK1) mediates the cellular effect of insulin and growth factors by activating a group of kinases including PKB/Akt, S6K, RSK, SGK and PKC isoforms. PDK1 possesses two regulatory domains namely a Pleckstrin Homology (PH) domain that binds to the phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] second messenger, and a substrate binding site termed the PIF-pocket. Employing a combination of biochemical, structural and mouse knock-in approaches we have been able to define the roles that the regulatory domains on PDK1 play. We have established that binding of PDK1 to PtdIns(3,4,5)P3 is essential for efficient activation of PKB isoforms as well as for maintaining normal cell size and insulin sensitivity. In contrast, the PIF-substrate binding pocket of PDK1 is not required for PKB activation, but is necessary for PDK1 to activate all of its other substrates.     

Evidence that 3-phosphoinositide-dependent protein kinase-1 mediates phosphorylation of p70 S6 kinase in vivo at Thr-412 as well as Thr-252

Protein kinase B and p70 S6 kinase are members of the cyclic AMP-dependent/cyclic GMP-dependent/protein kinase C subfamily of protein kinases and are activated by a phosphatidylinositol 3-kinase-dependent pathway when cells are stimulated with insulin or growth factors. Both of these kinases are activated in cells by phosphorylation of a conserved residue in the kinase domain (Thr-308 of protein kinase B (PKB) and Thr-252 of p70 S6 kinase) and another conserved residue located C-terminal to the kinase domain (Ser-473 of PKB and Thr-412 of p70 S6 kinase). Thr-308 of PKBalpha and Thr-252 of p70 S6 kinase are phosphorylated by 3-phosphoinositide-dependent protein kinase-1 (PDK1) in vitro. Recent work has shown that PDK1 interacts with a region of protein kinase C-related kinase-2, termed the PDK1 interacting fragment (PIF). Interaction with PIF converts PDK1 from a form that phosphorylates PKB at Thr-308 alone to a species capable of phosphorylating Ser-473 as well as Thr-308. This suggests that PDK1 may be the enzyme that phosphorylates both residues in vivo. Here we demonstrate that PDK1 is capable of phosphorylating p70 S6 kinase at Thr-412 in vitro. We study the effect of PIF on the ability of PDK1 to phosphorylate p70 S6 kinase. Surprisingly, we find that PDK1 bound to PIF is no longer able to interact with or phosphorylate p70 S6 kinase in vitro at either Thr-252 or Thr-412. The expression of PIF in cells prevents insulin-like growth factor 1 from inducing the activation of the p70 S6 kinase and its phosphorylation at Thr-412. Overexpression of PDK1 in cells induces the phosphorylation of p70 S6 kinase at Thr-412 in unstimulated cells, and a catalytically inactive mutant of PDK1 prevents the phosphorylation of p70 S6K at Thr-412 in insulin-like growth factor 1-stimulated cells. These observations indicate that PDK1 regulates the activation of p70 S6 kinase and provides evidence that PDK1 mediates the phosphorylation of p70 S6 kinase at Thr-412.     

3-磷酸肌醇依赖性蛋白激酶1结构和功能

PDK1可调节AGC激酶家族中一些重要蛋白激酶。这些激酶包括蛋白激酶B（PKB／Akt）、p70核小体S6激酶（p70 ribosomal S6 kinase,S6K）、血清和糖皮质激素诱导激酶（SGK）和蛋白激酶C（PKC）等,它们在细胞代谢、生长、增殖和存活等生理过程中具有重要作用。因此,了解PDK1生物学特性可能对其调节的AGC激酶持续活化的癌症治疗具有一定推动作用。本文对PDK1的结构、遗传和生化特点进行了综述。     

Development of PCR-based markers for thermosensitive genetic male sterility gene tms3(t) in rice (Oryza sativa L.)

Development of simple and reliable PCR-based markers is an important component of marker-aided selection (MAS) activities for agronomically important genes in rice breeding. In order to develop PCR-based markers for a rice thermosensitive genetic male sterility gene tms3(t), located on chromosome 6, the nucleotide sequences of four linked RAPD markers OPF18(2600), OPAC3(640), OPB19(750) and OPM7(550) were used to design and synthesize several pairs of specific primers for PCR amplification of the genomic DNA of both the parents IR32364TGMS (sterile) and IR68 (fertile), involved in mapping this gene. For the RAPD marker OPF 18(2600), two pairs of specific primer pair combination from different positions of the sequence resulted in generation of two codominant STS (Sequence Tagged Sites) markers. In case of markers OPAC3(640), OPB19(750) and OPAA7(550) the first two could generate dominant polymorphism, while the last one could not be successful in PCR amplification. Both the codominant STSs with primer combinations F18F/F18RM and F18FM/F18RM were found to be tightly linked to the tms3(t) gene with a genetic distance of 2.7 cM. The sizes of the different alleles in case of F18F/F18RM, F18FM/F18RM combinations were 2300 bp, 1050 bp, and 1900 bp, 1000 bp respectively. The efficiency of marker-assisted selection for this trait was estimated as 84.6%. Polymorphism survey of 12 elite rice lines, indicated that these PCR-based markers for tms3(t) can now be used in selecting TGMS plants at seeding stage in the segregating populations in environment independent of controlled temperature regime.     

Antisense display--a method for functional gene screening: evaluation in a cell-free system and isolation of angiogenesis-related genes.

Presented here is an antisense-oriented method for functional gene screening, which we propose naming 'antisense display'. In principle, it consists of four steps: (i) preparation of phosphorothioate antisense repertoires that would correspond to the Kozak's consensus sequence, (ii) subgroup screening to identify active antisense molecules that could cause changes in the cellular phenotypes concerned and (iii) RT-PCR cloning of cDNA with the 5[prime] sense complement and 3[prime] anchor primers and sequence determination, followed by (iv) functional assays of candidate genes. Cell-free translation in rabbit reticulocyte lysate revealed that 10mer or longer antisense effectively halted protein synthesis. This required the presence of RNase H, and was achieved without prior heat-denaturation of the RNA templates. Then, subpools of the 10mer repertoire were administered to human microvascular endothelial cells in culture, and screened for anti-angiogenic activities. A single species having the sequence 5[prime]-GGCTCATGGT-3[prime] consistently inhibited the endothelial cell growth under hypoxia. Through RT-PCR with the corresponding sense primer, we came across three candidate cDNAs. Experiments employing longer unique antisense reproduced marked growth inhibitions in two of the three cDNAs. One encoded a mitochondrial protein and the other, which encoded a putative type-2 membrane protein containing Rab-GAP/TBC and EF-hand like domains, was a gene previously undescribed in human. The results suggest that the antisense display method is potentially useful for isolating new genes towards elucidating their functions.     

Peroxovanadate induces tyrosine phosphorylation of phosphoinositide-dependent protein kinase-1 potential involvement of src kinase.

Phosphoinositide-dependent protein kinase-1 (PDK1) is a recently identified kinase that phosphorylates and activates protein kinase B (PKB). Activation of PKB by insulin is linked to its translocation from the cytosol to the plasma membrane. However, no data are available yet concerning the localization of PDK1 in insulin-sensitive tissue. Using isolated adipocytes, we studied the effect of insulin and of an insulin-mimicking agent peroxovanadate on the subcellular localization of PDK1. In unstimulated adipocytes, overexpressed PDK1 was mostly cytosolic with a low amount associated to membranes. Peroxovanadate stimulation induced the redistribution of PDK1 to the membranes while insulin was without effect. This peroxovanadate effect was dependent on phosphatidylinositol 3,4,5 triphosphate [PtdIns(3,4,5)P3] production as inhibition of PtdIns 3-kinase by wortmannin or deletion of the PH domain of PDK1 prevented the peroxovanadate-induced translocation of PDK1. Further, peroxovanadate-treatment induced a tyrosine phosphorylation of PDK1 which was wortmannin insensitive and did not require the PH domain of PDK1. An inhibitor of Src kinase (PP2) decreased the peroxovanadate-induced PDK1 tyrosine phosphorylation and overexpression of v-Src stimulated this phosphorylation. Mutation of tyrosine 373 of PDK1 abolished the v-Src induced PDK1 tyrosine phosphorylation and partially reduced the effect of peroxovanadate. Our findings suggest that PDK1 could be a substrate for tyrosine kinases and identify Src kinase as one of the tyrosine kinases able to phosphorylate PDK1.     

A novel real-time quantitative PCR method using attached universal template probe

A novel real-time quantitative polymerase chain reaction (PCR) method using an attached universal template (UT) probe is described. The UT is an approximately 20 base attachment to the 5′ end of a PCR primer, and it can hybridize with a complementary TaqMan probe. One of the advantages of this method is that different target DNA sequences can be detected employing the same UT probe, which substantially reduces the cost of real-time PCR set-up. In addition, this method could be used for simultaneous detection using a 6-carboxy-fluorescein-labeled UT probe for the target gene and a 5-hexachloro-fluorescein-labeled UT probe for the reference gene in a multiplex reaction. Moreover, the requirement of target DNA length for UT–PCR analysis is relatively flexible, and it could be as short as 56 bp in this report, suggesting the possibility of detecting target DNA from partially degraded samples. The UT–PCR system with degenerate primers could also be designed to screen homologous genes. Taken together, our results suggest that the UT–PCR technique is efficient, reliable, inexpensive and less labor-intensive for quantitative PCR analysis.