Sanger Sequencing for BRCA1 c.68_69del, BRCA1 c.5266dup and BRCA2 c.5946del Mutation Screen on Pap Smear Cytology Samples.

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.

A novel low temperature PCR assured high-fidelity DNA amplification.

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.

Identification of a NodD repressible gene adjacent to nodM in Rhizobium leguminosarum biovar viciae.

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.

Prevent cervical cancer by screening with reliable human papillomavirus detection and genotyping.

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 properties of NodD were affected by mere variation in length within its hinge 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 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 autoregulating 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.

Post-transcriptional regulation of NifA expression by Hfq and RNase E complex in Rhizobium leguminosarum bv. viciae.

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 Hfq-dependent 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'-untranslated region was that the cleavage made ribosome-binding sites accessible for translation.

A small functional intramolecular region of NodD was identified by mutation.

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.

Evidences of Hfq associates with tryptophanase and affects extracellular indole levels.

In this study, we observed a novel property of Escherichia coli Hfq protein: it possibly influenced extracellular indole levels. The extracellular indole concentrations were increased in Hfq mutant cells and decreased in Hfq overexpression cells in a cell density-dependent manner. The decreased extracellular indole levels in Hfq overexpression cells caused the postponement of entering into stationary phase. Indole was produced by tryptophanase, the gene product of tnaA, which catalyzed tryptophan into indole, ammonia and pyruvate. Further studies showed that at cell density of 0.8 but not at 0.4, tryptophanase activities of total cell extracts were affected by Hfq mutation or overexpression. Protein pull-down assay and co-immunoprecipitation experiments revealed that Hfq associated with tryptophanase under relatively higher extracellular indole levels, suggesting this was a feedback control of indole production. The association of Hfq and tryptophanase might be indirect because purified Hfq could not affect the values of Km and Vmax of purified tryptophanase.

Symbiotic plasmid is required for NolR to fully repress nodulation genes in Rhizobium leguminosarum A34.

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.

Site-specific fluorescent labeling approaches for naringenin, an essential flavonone in plant nitrogen-fixation signaling pathways.

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.

In vitro observation of the molecular interaction between NodD and its inducer naringenin as monitored by fluorescence resonance energy transfer.

At initial stages in the Rhizobium legume symbiosis, most nodulation 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.

Detecting AFP mRNA in peripheral blood of the patients with hepatocellular carcinoma, liver cirrhosis and hepatitis.

BACKGROUND: The low frequency of disseminated carcinoma cells in the blood now makes immunomagnetic bead sorting and reverse transcriptase-polymerase chain reaction (RT-PCR) technique more popular. METHODS: Three milliliters of peripheral blood were collected from 91 patients and 18 normal donors. The circulating carcinoma cells were enriched with CD45 and Ber-EP4 immunomagnetic beads. The alpha-fetoprotein (AFP) mRNA was amplified with nested RT-PCR. RESULTS: The total positive detection rate was 72.1%, 43.8%, 25.0%, 100%, and 66.7% in patients with hepatocellular carcinoma (HCC) untreated, liver cirrhosis (LC), hepatitis, metastasis liver cancer, and postsurgery of hepatocellular carcinoma, respectively. There was a significant difference among the patients with HCC, LC and hepatitis (HCC vs. LC, P<0.05; HCC vs. hepatitis, P<0.01) and between Class A and B of the HCC patients (P<0.05). Meanwhile, AFP mRNA was markedly expressed in HCC patients compared to the patients with no HCC (LC and hepatitis). The levels of aspartate transaminase (AST) and gamma-glutamyltranspeptidase (GGT) were significantly different in AFP mRNA-positive patients with autoimmune chronic active hepatitis B (CAHB) or LC in contrast to the corresponding negative patients. CONCLUSION: Combining negative and positive immunomagnetic bead sorting and RT-PCR technique can effectively detect circulating tumor cells. AFP mRNA is a more reliable marker of metastasis compared to serum AFP.

Modulating DNA bending affects NodD-mediated transcriptional control in Rhizobium leguminosarum.

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.

[The structural, transcriptional and homology analysis of two frr genes in rice].

Two rice (Oryza sativa subsp. japonica cv. Nipponbare) ribosome recycling factor genes--OsfrrA and OsfrrB had been identified and characterized in this study. The gene OsfrrA is located on chromosome 4 while OsfrrB on chromosome 7. No other homologue is found in rice organelle genomes. Both genes are unique in rice genome and constitutively expressed. The N-terminal character of their encoded protein products suggests that the proteins are transferred to mitochondrion and chloroplast respectively and carry out their functions. The sequence conservation and the constitutive expression profile of the two genes strongly imply their indispensable role in plant growth. In addition, these sequences share phylogenetic homology to some extent with other prokaryotic and eukaryotic RRFs, providing further evidence for the endosymbiotic theory, and implying the potential value of RRFs in molecular evolution research.

[Identification and analysis of a group of highly conserved trs-like genes in rice].

There are at least ten transcriptional trs-like genes in rice that have been confirmed by RT - PCR and sequencing, based on the annotation results of rice genome and homologous search. These ten genes correspond to six of the ten known subunits of TRAPP complex in yeast. Four pairs of them are duplicates while the other two are unique according to the known rice genomic sequences. All of the ten genes are constitutively expressed in rice tissues and share phylogenetic homology to some extent with other eukaryotic trs-like genes in their gene structures and protein sequences.

A HU-like gene mutation in Rhizobium leguminosarum bv. viciae affects the expression of nodulation genes.

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.

Transcript abundance of rml1, encoding a putative GT1-like factor in rice, is up-regulated by Magnaporthe grisea and down-regulated by light.

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.

[Characterization of the copy number of RIRE10 retrotransposon and transcriptional activity of its LTR in rice genome].

A full-length Ty3-like retrotransposon, named RIRE10, was identified on the long arm of chromosome 4 in rice genome. The internal region between two LTRs had another open reading frame in the region upstream of gag-pol sequence. The transcripts from LTR region were detected by Northern blot hybridization and RT-PCR. To assess the activity of RIRE10 in rice genome, the copy number of its internal region and long terminal repeat (LTR) domain were determined by dot blot analyses. Nearly 900 solo-LTR of the RIRE10 retrotransposon exist in rice genome, apart from those LTRs that flank 65 intact RIRE retrotransposons. Based on the experimental results, the retrotransposition of RIRE10 was speculated to be influenced by two factors: transcriptional activity of LTR region and homologous recombination resulting in solo-LTR.

Inactivation of the nod box distal half-site allows tetrameric NodD to activate nodA transcription in an inducer-independent manner.

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.

Structural analysis of a gene cluster encoding DFR-like proteins from rice chromosome 4.

Sequencing analysis of the 323 kb contig of rice chromosome 4 identified a gene cluster encoding 7 dihydroflavonol-4-reductase (DFR)-like proteins within a 56 kb region. The 7 DFR-like genes were found to be arranged in a tandem array, and all of them comprised 6 exons and 5 introns. Analysis of the predicted amino acid sequences demonstrated that these 7 proteins shared strong similarities with DFR and other enzymes of the phenylpropanoid biosynthesis pathway. RT-PCR revealed the expression pattern of the 7 genes was different in various rice tissues. The structural and functional features of these 7 DFR-like genes and their evolutionary implications are discussed.