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.

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.

[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.

[In vitro transcription of inducible nod gene nodA].

We observed in vitro transcription of nodA, an inducible nod gene. Primer extension analysis of the transcripts showed there always existed a transcription product identical to that of its in vivo transcription, when 10 mumol/L naringening and NodD protein was added in the in vitro transcription system. Induced in vitro transcription of nodA gene suggests that in its in vivo transcription there may also exist a kind of physical interaction among NodD protein, inducer naringening and promoter of nodA gene.

Identification and Structural Analysis of a Class of Potentially Transposable Solo-LTR in Rice Genome.

Two solo-LTRs, named SLTR1 and SLTR2, were found in BAC t17804 and q5343 on rice chromosome 4, respectively. SLTR1 is in a 18 S rRNA gene and SLTR2 is in a retrotransposon. They share sequence homology and show sequence similarity 89.1% and 70.1% to the LTR of rice retrotransposon RIRE8, respectively. SLTR1 and SLTR2 are of gypsy retrotransposons of rice. They are both highly repetitive sequences and widely distributed in the rice genome, as shown by hybridization with specific probes of SLTR1 and SLTR2. Using PCR amplication with primers on flanking sequences of SLTR1 and SLTR2, no bands corresponding to those of BACs were amplified using the rice genomic DNA as template. SLTR1 and SLTR2 did not locate in the relative loci of the rice genome, as supported by hybridization with specific probes of genes interrupted by them. Obviously, SLTR1 and SLTR2 reported here came from different loci of the genome by the transposition. These solo-LTRs may be useful for our rice genome studies.

Structural Analysis of a Gene Cluster Encoding Two Cationic and Three Anionic Peroxidases from Rice Chromosome 4.

Sequence analysis of a rice BAC q3037(H0207F01) identified a cluster of five tandemly arranged peroxidase genes, osp1,osp2,osp3,osp4 and osp5, within a 22.5 kb region. osp4,osp5 each have three exons interrupted by two introns, while osp1,osp2 and osp3 each have two exons interrupted by a single intron. The five genes were predicted products of 338, 335, 336, 343 and 346 amino acid residues, respectively, including putative signal peptide sequence at the amino-termini. And OSP1, OSP4 and OSP5 were predicted to be anionic peroxidase, OSP2 and OSP3 are cationic. Comparative analysis and evolutionary analysis of the clustered genes and other peroxidase family members revealed that the gene cluster occurred by tandemly gene duplications(from osp5 to osp1) and that osp5,ap1 and prx7 were potential orthologies, and osp1-5, ap1 and prx7 constituted a novel evolutionary branch of class III peroxidases.

A Promoter Overlapping with that of nodF is Responsible for a New RNA Molecule Transcription in Rhizobium leguminosarum.

The expression of nod genes of rhizobia is controlled at the transcriptional level in a complicated way. In this work, a new promoter, which is responsible for an RNA molecule transcription in the opposite direction to that of nodF, was identified in Rhizobium leguminosarum. This new promoter was characterized to overlap with that of nodF, and the size of px(2), the RNA molecule initiated from this promoter was determined to be of approximately 0.72 kb in length. The study on px2 showed that its transcription depended on the Escherichia coli sigma(70)- like factor of Rhizobium leguminosarum, and that the in vitro transcription of px(2) was inhibited by Px, a newly identified regulatory protein in our lab. The involvement of px(2) in the regulation of nod gene expression is suggested.

Preferential Location of MITEs in Rice Genome.

To observe the association between MITEs and genes in details, 82 kb of the rice genomic DNA were sequenced, and 10 genes were identified by similarity search. It was found that miniature inverted-repeat transposable elements (MITEs) were located upstream of the genes identified without exception, regardless of their directions of transcription. One MITE was found to be located between two 5' regions of the genes with opposite orientation of transcription, indicating that it was shared by them. The positional patterns of MITEs may therefore serve as an aid for preliminary screening of genes.

Cloning, Expression, Characterization and Application of Bst DNA Polymerase Large Fragment.

DNA sequencing using thermostable DNA polymerase is very valuable in molecular biology. From a specific strain of Bacillus stearothermophilus, the gene of the Bst DNA polymerase large fragment which had no 5'-3' exonuclease activity was cloned and recombined into an expression vector pYZ23,and the constructed plasmid was introduced into Escherichia coli JF1125,then the expressed protein was isolated and purified. The genetic engineered product shows characteristics similar with the purified natural Bst DNA polymerase large fragment. It will benefit the wide application of Bst DNA polymerase and the study of the relationship between the structure and function of Bst DNA polymerase.