Search

As previously reported, a novel low temperature (LoTemp) polymerase chain reaction (PCR) catalyzed by a moderately heat-resistant (MHR) DNA polymerase with a chemical-assisted denaturation temperature set at 85 °C instead of the conventional 94–96 °C can achieve high-fidelity DNA amplification of a target DNA, even after up to 120 PCR thermal cycles. Furthermore, such accurate amplification is not achievable with conventional PCR. Now, using a well-recognized L1 gene segment of the human papillomavirus (HPV) type 52 (HPV-52) as the template for experiments, we demonstrate that the LoTemp high-fidelity DNA amplification is attributed to an unusually high processivity and stability of the MHR DNA polymerase whose high fidelity in template-directed DNA synthesis is independent of non-existent 3'–5' exonuclease activity. Further studies and understanding of the characteristics of the LoTemp PCR technology may facilitate implementation of DNA sequencing-based diagnostics at the point of care in community hospital laboratories.

The incidence of cervical cancer is expected to rise sharply in China. A reliable routine human papillomavirus (HPV) detection and genotyping test to be supplemented by the limited Papanicolaou cytology facilities is urgently needed to help identify the patients with cervical precancer for preventive interventions. To this end, we evaluated a nested polymerase chain reaction (PCR) protocol for detection of HPV L1 gene DNA in cervicovaginal cells. The PCR amplicons were genotyped by direct DNA sequencing. In parallel, split samples were subjected to a Digene HC2 HPV test which has been widely used for “cervical cancer risk” screen. Of the 1826 specimens, 1655 contained sufficient materials for analysis and 657 were truly negative. PCR/DNA sequencing showed 674 infected by a single high-risk HPV, 188 by a single low-risk HPV, and 136 by multiple HPV genotypes with up to five HPV genotypes in one specimen. In comparison, the HC2 test classified 713 specimens as infected by high-risk HPV, and 942 as negative for HPV infections. The high-risk HC2 test correctly detected 388 (57.6%) of the 674 high-risk HPV isolates in clinical specimens, mislabeled 88 (46.8%) of the 188 low-risk HPV isolates as high-risk genotypes, and classified 180 (27.4%) of the 657 “true-negative” samples as being infected by high-risk HPV. It was found to cross-react with 20 low-risk HPV genotypes. We conclude that nested PCR detection of HPV followed by short target DNA sequencing can be used for screening and genotyping to formulate a paradigm in clinical management of HPV-related disorders in a rapidly developing economy.

The nodFEL and nodMNT operons in Rhizobium leguminosarum biovar viciae are transcribed in the same orientation and induced by NodD in response to flavonoids secreted by legumes. In the narrow intergenic region between nodFEL and nodMNT, we identified a small gene divergently transcribed from nodM to the 3 end of nodL. Unlike the promoters upstream of nodF and nodM, the promoter of this gene is constitutively expressed. It appeared that its promoter might partially overlap with that of nodM and its expression was repressed by nodD. A deletion mutation was made and proteins produced by the mutant were compared with those by wild-type using 2D gel electrophoresis. Several protein differences were identified suggesting that this small gene influences the expression or stability of these proteins. However, the mutant nodulated its host plant (pea) normally.

In Rhizobium leguminosarum bv. viciae, NodD, as a member of the LysR-type transcriptional regulators (LTTRs), exerts auto-regulation and activates transcription of other nod genes in the presence of naringenin. LTTRs were typically composed of N-terminal DNA-binding domain and C-terminal regulatory domain. In this study, by systematic insertion mutation, a region of 12 amino acids in length of NodD was identified as functional domain. Insertion mutants in this region appeared to acquire the ability of constitutively activating nodA gene and retained their auto-regulation properties. This identified region was shown to be a hinge of NodD as revealed through the model built using Swiss- PDB Viewer software. It is the first time to report that as a member of LysR family, NodD has been shown to contain a short intramolecular domain that influences its performance.

NifA is the general transcriptional activator of nitrogen fixation genes in diazotrophic bacteria. In Rhizobium leguminosarum bv. viciae strain 8401/pRL1JI, the NifA gene is part of a gene cluster (fixABCXNifAB). In this study, results showed that in R. leguminosarum bv. viciae 8401/pRL1JI, host factor required (Hfq), and RNase E were involved in the post-transcriptional regulation of NifA expression. It was found that Hfqdependent RNase E cleavage of NifA mRNA was essential for NifA translation. The cleavage site is located at 32 nucleotides upstream of the NifA translational start codon. A possible explanation based on predicted RNA secondary structure of the NifA 5 0 -untranslated region was that the cleavage made ribosome-binding sites accessible for translation.

NolR is a regulator of nodulation genes present in Rhizobium and Sinorhizobium. However, the mechanism by which NolR participates in the inducible transcription of nodulation genes remains unclear. To investigate whether there are other factors regulating the function of NolR, an insertion mutant of NolR in Rhizobium leguminosarum strain 8401, which lacks the symbiotic plasmid, was constructed by homologous recombination. We investigated the effects of NolRinactivation on the expression of nodulation genes. Three inducible nodulation genes (nodA, nodF and nodM) were expressed constitutively in NolR mutant, MR114. Our results suggested that the symbiotic plasmid is required for NolR to fully repress nodulation genes in Rhizobium leguminosarum A34. In addition, MR114 has provided a useful tool for further study of molecular interactions between NolR and other factors.

At initial stages in the Rhizobium legume symbiosis, most Modulation genes are controlled by NodD protein and plant inducers. Some genetic studies and other reports have suggested that NodD may be activated by its direct interaction with plant inducers. However, there has been no molecular evidence of such an inducing interaction. In this paper, we used fluorescence resonance energy transfer technique to see whether such an interaction exists between NodD and its activator, naringenin, in vitro. The tetracysteine motif (Cys-Cys-Pro-Gly-Cys-Cys) was genetically inserted into NodD to label NodD with 4',5'-bis(1,3,2-dithioarsolan-2-yl) fluorescein (FlAsH). Naringenin was labeled with fluorescein by chemical linking. In the fluorescence resonance energy transfer experiments in vitro, the fluorescence intensity of one acceptor, NodD(90R6)-FlAsH, increased by 13%. This suggests that NodD may directly interact with inducer naringenin in vitro and that the reaction centre is likely near hinge region 1 of NodD.

Three sets of polymerase chain reaction (PCR) primers were designed for heminested PCR amplification of the target DNA fragments in the human genome which include the site of BRCA1 c.68_69del, BRCA1 c.5266dup and BRCA2 c.5946del respectively, to prepare the templates for direct Sanger sequencing screen of these three founder mutations. With a robust PCR mixture, crude proteinase K digestate of the fixed cervicovaginal cells in the liquid-based Papanicolaou (Pap) cytology specimens can be used as the sample for target DNA amplification without pre-PCR DNA extraction, purification and quantitation. The post-PCR products can be used directly as the sequencing templates without further purification or quantitation. By simplifying the frontend procedures for template preparation, the cost for screening these three founder mutations can be reduced to about US $200 per test when performed in conjunction with human papillomavirus (HPV) assays now routinely ordered for cervical cancer prevention. With this projected price structure, selective patients in a high-risk population can be tested and each provided with a set of DNA sequencing electropherograms to document the absence or presence of these founder mutations in her genome to help assess inherited susceptibility to breast and ovarian cancer in this era of precision molecular personalized medicine.

Rice, one of the world's most important food plants, has important syntenic relationships with the other cereal species and is a model plant for the grasses. Here we present a map-based, finished quality sequence that covers 95% of the 389 Mb genome, including virtually all of the euchromatin and two complete centromeres. A total of 37,544 non-transposable-element-related protein-coding genes were identified, of which 71% had a putative homologue in Arabidopsis. In a reciprocal analysis, 90% of the Arabidopsis proteins had a putative homologue in the predicted rice proteome. Twenty-nine per cent of the 37,544 predicted genes appear in clustered gene families. The number and classes of transposable elements found in the rice genome are consistent with the expansion of syntenic regions in the maize and sorghum genomes. We find evidence for widespread and recurrent gene transfer from the organelles to the nuclear chromosomes. The map-based sequence has proven useful for the identification of genes underlying agronomic traits. The additional single-nucleotide polymorphisms and simple sequence repeats identified in our study should accelerate improvements in rice production.

Rhizobium leguminosarum NodD binds to the nod box of the inducible nod gene nodA as a V-shaped tetramer and bends the nod box. In this work, we show that the nod gene inducer naringenin decreased gel mobility of nod box DNA–NodD complexes by sharpening the NodD-induced DNA bend, which correlated with nodA transcription activation. NodD can induce different DNA bends when the distance between the two half-sites of the nod box was modified, which severely affected NodD-mediated transcriptional control. One or two base pairs were deleted from, or inserted into, the two half-sites of the nod box of nodA. Circular permutation assays showed that such distance modulations allowed NodD to induce relaxed or sharpened DNA bending. In the case of 1 bp deletion, where the DNA bends were more relaxed than in the wild type, nodA transcription was repressed both in the absence and in the presence of inducer naringenin. In the cases of 1 and 2 bp insertion, where the DNA bends were much sharper than in wild type in the absence or presence of the inducer naringenin, nodA transcription was initiated constitutively with no requirement for the inducer naringenin or, even, the NodD regulating protein.

NodD is the major regulator of nod genes expression in rhizobia. Previously, a HU-like protein in Rhizobium leguminosarum bv. viciae has been identified to bind specifically with nod promoters and be involved in in vitro nodD transcription, but its in vivo function remained unknown. In this work we have cloned and sequenced the R. leguminosarum bv. viciae gene, named hurL, for this HU-like protein. Using the E. coli-expressed HurL proteins, we proved that HurL had high affinity to several nod promoters and showed a stimulation effect on in vitro nodD transcription at appropriate concentration. The R. leguminosarum bv. viciae hurL gene was mutated by insertion of a kanamycin resistance cassette. The obtained hurL mutant strain M704 exhibited poor growth under free-living conditions and failed to induce nodules on Pisum sativum cv. Frisson and Vicia hirsuta. Further studies of NodD production and nod genes-lacZ fusions expression in the hurL mutant revealed that inactivation of hurL led to severe impairment in the nodD expression, repression in the inducible expression of nodA and nodF, and slight enhancement in the expression of px2, a gene identified earlier in this lab. These results suggested that hurL might be required for maintaining the normal expression of nod genes in R. leguminosarum bv. viciae.

In Rhizobium leguminosarum, NodD can activate nodA transcription in response to inducer flavonoids. Here, we show that the inducible nodA promoter contains an intrinsic part through which NodD can activate nodA transcription in an inducer-independent manner. Evidence was provided that NodD binds to target DNA through anchoring the two half-sites of the nod box as a tetramer. An imperfect inverted repeat AT-N10-GAT was found in each half-site and is critical for NodD binding. Mutation of the inverted repeat of the nod box distal half-site allowed NodD to activate nodA transcription in an inducer-independent manner in vivo, and to modulate the DNA bending of the NodD-nod box complex in the absence of inducer in vitro.

Rice is the principal food for over half of the population of the world. With its genome size of 430 megabase pairs (Mb), the cultivated rice species Oryza sativa is a model plant for genome research. Here we report the sequence analysis of chromosome 4 of O. sativa, one of the first two rice chromosomes to be sequenced completely. The finished sequence spans 34.6 Mb and represents 97.3% of the chromosome. In addition, we report the longest known sequence for a plant centromere, a completely sequenced contig of 1.16 Mb corresponding to the centromeric region of chromosome 4. We predict 4,658 protein coding genes and 70 transfer RNA genes. A total of 1,681 predicted genes match available unique rice expressed sequence tags. Transposable elements have a pronounced bias towards the euchromatic regions, indicating a close correlation of their distributions to genes along the chromosome. Comparative genome analysis between cultivated rice subspecies shows that there is an overall syntenic relationship between the chromosomes and divergence at the level of single-nucleotide polymorphisms and insertions and deletions. By contrast, there is little conservation in gene order between rice and Arabidopsis.

A Rhizobium expression system was constructed via introducing strong transcriptional elements to an IncP broad host range plasmid pKT230. Using this system, the nodD gene of Rhizobium leguminosarum biovar viciae was overexpressed in its own in vivo environment. Western blot showed that the NodD yield of the newly constructed expression vector pKNDT was much higher than that of a reported vector pIJ1518. Based on the result from quantitative gel retardation assay, the specific DNA-binding activity of NodD was estimated to increase by about 80-fold. Purification of NodD was performed by the sequential steps: polyethyleneimine treatment, ammonium sulfate fractional precipitation, and cellulose phosphate P11 chromatography. About 26 mg NodD with a purity about 90% was obtained from 14 g wet weight cell pellet within 2 days.

In many flowering plants, self-fertilization is prevented by an intraspecific reproductive barrier known as self-incompatibility (SI), that, in most cases, is controlled by a single multiallelic S locus. So far, the only known S locus product in self-incompatible species from the Solanaceae, Scrophulariaceae and Rosaceae is a class of ribonucleases called S RNases. Molecular and transgenic analyses have shown that S RNases are responsible for pollen rejection by the pistil but have no role in pollen expression of SI, which appears to be mediated by a gene called the pollen self-incompatibility or Sp gene. To identify possible candidates for this gene, we investigated the genomic structure of the S locus in Antirrhinum, a member of the Scrophulariaceae. A novel F-box gene, AhSLF-S2, encoded by the S2 allele, with the expected features of the Sp gene was identified. AhSLF-S2 is located 9 kb downstream of S2 RNase gene and encodes a polypeptide of 376 amino acids with a conserved F-box domain in its amino-terminal part. Hypothetical genes homologous to AhSLF-S2 are apparent in the sequenced genomic DNA of Arabidopsis and rice. Together, they define a large gene family, named SLF (S locus F-box) family. AhSLF-S2 is highly polymorphic and is specifically expressed in tapetum, microspores and pollen grains in an allele-specific manner. The possibility that Sp encodes an F-box protein and the implications of this for the operation of self-incompatibility are discussed.

A 2225 bp cDNA, designated RPA1, was isolated from an Oryza sativa cDNA library. Analysis revealed a 1761 bp coding sequence with 15 non-identical repeat units. The ORF encoded the A regulatory subunit of protein phosphatase 2A (PP2A-A) as ascertained by complementation of the yeast tpd3 mutant defective in this gene. The corresponding genomic DNA from a rice genome BAC library revealed that the gene contains eleven introns. The rice genome contains only a single copy of this gene as judged by Southern blot analysis. The PP2A protein is highly conserved in nature; the rice protein shows 88% amino acid identity with its counterparts in Arabidopsis or Nicotiana tabacum.

nodFEL operon is one of the NodD-dependent inducible nod operons that is clustered on the symbiosis plasmid of Rhizobium leguminosarum biovar viciae. A recent study on the upstream regulatory region of nodFEL operon had identified a new promoter, which was responsible for the transcription of a 0.72 Kb RNA molecule, called px(2), in the opposite direction to nodF. This new promoter was further characterized to overlap with that of nodF and its in vitro transcription was inhibited by another newly identified nod regulator, Px. In this paper we report that the sequence analysis of the px(2) transcript revealed only one open reading frame (ORF(66)), corresponding to a polypeptide of 66 amino acids. Moreover, the increase in px(2) copy numbers enhanced the in vivo inducible expression level of nodF, whereas the frame-shift mutation of ORF(66) eliminated such effect, providing evidence that px(2) is responsible for specifically upregulating nodF expression. This result also raises the big possibility that px(2) encodes this polypeptide. A model for coordinated expression of px(2) and nodF, transcribed divergently from each other, is proposed.

The nodFEL and nodMNT operons in Rhizobium leguminosarum biovar viciae are transcribed in the same orientation and induced by NodD in response to flavonoids secreted by legumes. In the narrow intergenic region between nodFEL and nodMNT, we identified a small gene divergently transcribed from nodM to the 3' end of nodL. Unlike the promoters upstream of nodF and nodM, the promoter of this gene is constitutively expressed. It appeared that its promoter might partially overlap with that of nodM and its expression was repressed by nodD. A deletion mutation was made and proteins produced by the mutant were compared with those by wild-type using 2D gel electrophoresis. Several protein differences were identified suggesting that this small gene influences the expression or stability of these proteins. However, the mutant nodulated its host plant (pea) normally.

Construction of a full bacterial artificial chromosome (BAC) library of Oryza sativa genome

In Rhizobium leguminosarum (R.l.) biovar viciae, the nodulation gene nodD encodes a transcriptional activator (NodD) which binds to highly conserved DNA sequences (nod-boxes) in the promoters of other nod operons. In addition, NodD represses nodD transcription and this occurs at the divergent and overlapping nodA-nodD promoters. We mutagenised this region with hydroxylamine, and by cloning the mutagenised DNA into a vector carrying the lacZ reporter gene downstream from the cloning site identified mutations affecting nodD expression and repression. The resulting plasmids were transferred to R. l. viciae strains containing or lacking nodD. Two classes of promoter mutants were identified: those in which nodD transcription was altered and those in which NodD-dependent repression was altered. The nucleotide (nt) changes in the promoter region were found to be located within two inverted repeat sequences (A2 and A3) which are about 70 bp apart. A2 is important for nodD transcription and A3 (which is upstream from A2) is involved in NodD-dependent repression. The nt sequence at A3 shows some homology to the nod-box region of the nodA promoter. It is proposed that the NodD-dependent repression occurs as a result of NodD binding to both A3 and the nodA nod-box, forming a loop which prevents transcription of nodD from its promoter, A2, which lies between A3 and the nod-box. This model is supported by the observation that there are at least three sites for NodD binding in the nodA-nodD promoter region.

In Rhizobium leguminosarum bv. viciae, NodD, a member of the LysR-type transcriptional regulators, while auto-regulating, activates transcription of other nod genes in the presence of naringenin. A hinge region of NodD was previously identified in our laboratory as a functional region independent of its N-terminal DNA-binding and C-terminal regulatory domain. Further study was carried out to see the possible effect of the length variation in the hinge region on NodD's properties. To our surprise, as many as seven classes of phenotypes were observed. Class I is deficient of activating nodA transcription and abolishes auto-regulation; class II is able to activate nodA transcription independently of naringenin and abolishes auto-regulation; class III retains auto-regulating but partial activating ability; class IV is able to activate transcription independently of naringenin and retains auto-regulation; in class V, nodA is transcribed constitutively but the transcription level is drastically down-regulated in the presence of naringenin; in class VI, nodA is transcribed constitutively with higher induction ratio; in class VII, nodA is transcribed constitutively with lower induction ratio. To learn more about the possible mechanism, circular permutation assays were done, which showed that the length variation of the hinge of NodD caused by mutation led to the change in bend angles of nod promoter. This finding should help to get an insight into how transcriptional regulation is mediated by NodD at the molecular level as well as to understand the regulatory system of this important family.

NolR is a regulator of nodulation genes present in Rhizobium and Sinorhizobium. However, the mechanism by which NolR participates in the inducible transcription of nodulation genes remains unclear. To investigate whether there are other factors regulating the function of NolR, an insertion mutant of NolR in Rhizobium leguminosarum strain 8401, which lacks the symbiotic plasmid, was constructed by homologous recombination. We investigated the effects of NolR inactivation on the expression of nodulation genes. Three inducible nodulation genes (nodA, nodF and nodM) were expressed constitutively in NolR- mutant, MR114. Our results suggested that the symbiotic plasmid is required for NolR to fully repress nodulation genes in Rhizobium leguminosarum A34. In addition, MR114 has provided a useful tool for further study of molecular interactions between NolR and other factors.

In search of an appropriate position for the fluorescent labeling, six chemically available positions of the flavonone core of naringenin have been examined. A number of azido-containing naringenin derivatives were accordingly prepared in various site-specific fashions, and the mild Cu(I)-catalyzed Huisgen 1,3-dipolar cycloaddition successfully served as the common "Click" labeling tool in the final steps. On the basis of the biological activities of the first batch of labeled compounds, further optimization at the C-6 position of naringenin finally afforded naringenin-flu (27), which acquired 20% of the potency of naringenin and presented good optical properties. Entry of naringenin-flu into living Rhizobium cells was demonstrated by in vitro fluorescent imaging experiments.

We isolated and sequenced both genomic DNA and cDNA clones, which encoded a putative GT1-like protein with 385 amino acids, from cultivated rice (Oryza sativa ssp. indica). This protein shows significant amino acid sequence similarities with trihelix DNA-binding GT-1a/B2F and GT-1 factors that were identified in dicot plants. Northern blotting analysis indicated that the transcript of the rice GT-1 factor in seedling was up-regulated by the rice blast fungus Magnaporthe grisea, down-regulated by various continuous light conditions and expressed rhythmically in light/dark cycles. This GT1-like factor gene was therefore designated as rml1 (rice gene regulated by M. grisea and light). The putative RML1 protein, encoded by this single copy gene, is thus identified as a new member of the plant-specific GT family of transcription factors in rice.

In many flowering plants, self-fertilization is prevented by an intraspecific reproductive barrier known as self-incompatibility (SI), that, in most cases, is controlled by a single multiallelic S locus. So far, the only known S locus product in self-incompatible species from the Solanaceae, Scrophulariaceae and Rosaceae is a class of ribonucleases called S RNases. Molecular and transgenic analyses have shown that S RNases are responsible for pollen rejection by the pistil but have no role in pollen expression of SI, which appears to be mediated by a gene called the pollen self-incompatibility or Sp gene. To identify possible candidates for this gene, we investigated the genomic structure of the S locus in Antirrhinum, a member of the Scrophulariaceae. A novel F-box gene, AhSLF-S2, encoded by the S2 allele, with the expected features of the Sp gene was identified. AhSLF-S2 is located 9 kb downstream of S2 RNase gene and encodes a polypeptide of 376 amino acids with a conserved F-box domain in its amino-terminal part. Hypothetical genes homologous to AhSLF-S2 are apparent in the sequenced genomic DNA of Arabidopsis and rice. Together, they define a large gene family, named SLF (S locus F-box) family. AhSLF-S2 is highly polymorphic and is specifically expressed in tapetum, microspores and pollen grains in an allele-specific manner. The possibility that Sp encodes an F-box protein and the implications of this for the operation of self-incompatibility are discussed.

We isolated RNA polymerase (RNAP) from Rhizobium leguminosarum, the nitrogen-fixing symbiont of peas and VICIA: Its 91 kDa subunit, which is homologous to sigma(70) of Escherichia coli RNAP, is necessary for transcription of the regulatory nodD gene, which in the presence of certain flavonoids induces transcription of other nod genes that are needed for the early steps of infection. We also show that negative autoregulation of nodD was achieved through competition between RNAP and NodD for their respective binding sites, which largely overlap. Combined with the result that high concentrations of the flavonoid inducer naringenin relieved the binding affinity of NodD for its target DNA, the way in which the nod genes are activated is discussed.

92% of the rice genome (4.3 x 10(8) bp, 2n = 24) was covered by 631 contigs of various length, which were generated by fingerprinting from a representative and genetically stable bacterial artificial chromosome (BAC) library of the Guang Lu Ai 4 (a O. Sativa variety) genome with the average insert of 120 kb in length. To form the contig map, 565 molecular markers of RFLP, STS, cDNA and anchor set derived from two O. Sativa varieties were by colony hybridization mapped to the contigs, which were then assigned to and ordered along the particular chromosomes according to the marker colinearity. Being highly conserved DNA sequences shared among the genomes of rice, barley, wheat, oat, maize, sorghum and sugar cane, 89 anchor markers mapped help to identify the rice genes through the information provided by the maps of relative genomes, and vice versa. Numerous repeated DNA sequences of various length were identified and mapped to the chromosomes. Physical distances have been determined for hundreds pairs of adjacent markers, which would facilitate the identification by map-based cloning the rice genes of interest. The accuracy of clone overlaps in contigs was further confirmed by the existence in contigs of well fit stacks of marker-lodged clones independently identified by hybridization. Large scale DNA sequencing of individual chromosomes could now be initiated simply by selecting and sequencing the minimally overlapped BAC clones of the contigs.

A rapid and accurate strategy for rice contig map construction was described. Rice BAC library with average insert of 120 kb in length was used as building materials in contig mapping. The contigs of varied lengths ranging from 500 kb to several megabases with sufficient redundancy to ensure the accuracy of the joining between individual BACs were formed by fingerprinting. The contigs were then assigned to and ordered along the chromosomes by various molecular markers through their hybridization against the whole rice genomic library. The accuracy of clone overlaps in contig was further confirmed by the existence in contigs of well fit stacks of marker-lodged clones. He contigs thus obtained covered nearly the rice genome.