Clinical significance of serum EGFR gene mutation and serum tumor markers in predicting tyrosine kinase inhibitor efficacy in lung adenocarcinoma.

OBJECTIVE: To study the clinical significance of serum epidermal growth factor receptor (EGFR) gene mutation and serum tumor markers in the prediction of tyrosine kinase inhibitor (TKI) efficacy in patients with lung adenocarcinoma. METHODS: Ninety patients with pathologically diagnosed lung adenocarcinoma were enrolled. Further, 51 out of 90 patients received the EGFR-TKI therapy, oral gefitinib. The correlations among serum EGFR gene mutations in exons 18-21, serum tumor markers such as carcinoembryonic antigen (CEA), carbohydrate antigen 24-2 (CA24-2), carbohydrate antigen 125, carbohydrate antigen 15-3 as well as carbohydrate antigen 19-9 (CA19-9) levels, and EGFR-TKI efficacy were determined. RESULTS: There was a high consistency of EGFR gene mutation rate between serum and tissue samples. The serum EGFR gene mutation rate in female patients or non-smokers was significantly higher than that in male patients or smokers, respectively. Serum CA19-9, CA24-2, and CEA levels were significantly correlated with serum EGFR mutation. After receiving gefitinib, the progression-free survivals (PFSs) of patients with high serum CEA level, high serum CA19-9 level, or serum EGFR gene mutation were significantly higher than those of normal patients, respectively. The PFSs were significantly prolonged in patients with EGFR gene mutation and high serum CEA level or patients with EGFR gene mutation and high serum CA19-9 level compared with those in patients with one abnormal biomarker and normal patients. CONCLUSION: Combined detection of EGFR gene mutations as well as CA19-9 and CEA levels in peripheral blood can predict the efficacy of EGFR-TKI in the treatment of patients with lung adenocarcinoma.

[A genetically modified japonica restorer line, C418-Xa21, and its hybrid rice with bacterial blight resistance].

The cloned bacterial blight (BB) resistance gene Xa21 was transferred into C418, a major restorer line of japonica hybrid rice in China, using an Agrobacterium-mediated system. The integrated single copy of transgene displayed a 3:1 segregation ratio in T1 generation in PCR and resistance analyses. The transgenic homozygous C418-Xa21 lines were selected in T2 generation through PCR and resistance analyses. The selected transgenic restorer lines were then crossed with a commonly used sterile line, TijinA, to produce Xa21 transgenic hybrid rice. Molecular analysis revealed that the produced hybrid rice, named as Tiyou418-Xa21, inherited the transgene. Both C418-Xa21 and Tiyou418-Xa21 plants displayed high resistance with a broad spectrum to Xoo races and maintained their normal elite agronomic characters. We also observed that the resistance level of Tiyou418-Xa21 was obviously higher than that of C418-Xa21 which may be attributed to their differences in genetic background. The propagation of this BB resistant hybrid variety with the transgene Xa21 with extend hybrid rice production in north China.

[Amplification and analysis of T-DNA flanking sequences in transgenic rice].

Rice transformation mediated by Agrobacterium tumefaciens has technically matured to some extent, but the mechanics of T-DNA integration in transgenic rice remains largely unknown. Using thermal asymmetric interlaced PCR (TAIL-PCR), we analyzed the flanking sequences of T-DNAs in transgenic rice plants, in which the resistance gene for rice bacterial blight disease, Xa21, had been integrated stably. Sequence analysis of 24 fragments amplified by TAIL-PCR showed that of them 14 were rice genomic DNA, 9 contained vector backbone sequences, and one was a fragment of the exogenous gene Xa21. The characteristics of the 14 rice genomic DNA sequences at T-DNA integrated sites are significantly different from those of the reported rice genomic sequences into which exogenous genes were integrated through direct DNA transformation methods. T-DNA border sequences integrated in rice genome have similar features as those integrated in dicotyledonous genomes. In the backbone containing flanking sequences (37.5%, 9/24) vector backbones appeared in different types.

[Construction of a bacterial artificial chromosome (BAC) contig encompassing the bacterial blight resistance gene Xa4 locus in rice].

The gene Xa4 confers dominantly resistance to rice bacterial blight, which has been finely mapped between RFLP markers G181 and L1044, and co-segregated with the resistance gene homologues sequence marker RS13. The three markers were used to screen a rice Bacterial Artificial Chromosome (BAC) library constructed from IRBB56, a Xa4-harborring indica variety, resulting in the detection of totally 128 positive clones. Of the 18 positive clones picked out by RS13, 4 and 6 clones were simultaneously detected by G181 and L1044, respectively. Based on their HindIII restriction patterns, 12 clones were selected out to construct a contig that spanned about 420 kb covering the Xa4 locus, which is a solid base for the isolation of Xa4 gene.

[Construction of a deep coverage rice BAC library and identification of clones associated with disease-resistant genes].

A BAC library for IRBB56, an accession pyramiding Xa4, xa5 and xa13 three bacterial blight resistance genes, was constructed. The library contains 55,296 clones with an average insert size of 132 kb. Based on a haploid genome size of 450 Mb, the coverage of the library was about 14 genome equivalents that make it one of the most comprehensive BAC libraries available in rice and provide 99.99% possibility to isolate any interested rice genes or sequences in the library. To determine the representation of organelle DNA homologues in the library, the library was screened with three different chloroplast genes and four mitochondrial genes, respectively. Results from this screening showed that less than 1% of clones in the library contain organelle genomic DNA homologues. Then, DNA markers on three different chromosomes linked to Xa4, xa5, and xa13, and a PCR fragment of rice UROD gene, were used to screen the library resulting in a range of 11-106 hits that will promote the isolation of these genes. The deep coverage and the large insert size of the library will facilitate physical mapping, isolation, and cloning of rice genes.

[Introduction of wide spectrum rice bacterial blight resistance gene Xa21 into two-line genic male sterile rice variety Pei'ai 64S].

Agrobacterium-mediated transformation of two-line genic male sterile Indica rice variety Pei'ai 64S was conducted using a cloned gene, Xa21, as the foreign gene and mature embryo calli as the recipients. A total of 46 transgenic plants had been obtained. The PCR analysis and Southern blotting showed the integration of Xa21 gene into the genome the transgenic plants. Results of inoculation with philippine race 6 of Xanthomonas oryzae pv. oryzae indicated that most of transgenic plants obtained high resistance to rice bacterial blight disease (Xoo). Analyses of T1 plants of the tested transgenic lines showed that integrated Xa21 gene could be steadily inherited and segregated in a 3:1 ratio.

[Integration of the genetic map and the physical map of the subterminal region on the longer arm of rice chromosome 6].

The region between RFLP markers G342 and R1167 was the subterminal part of the longer arm on the rice chromosome 6, because Shen et al. (1998) mapped two telomeric repeat associated sequences distal to G342. In order to integrate the genetic map and the physical map of the region, G342 and R1167 were firstly used to screen BAC library. Based on the positive clones detected by the two markers and chromosome walking by using the outer most insert-end of the overlapping clones, a contig containing 16 BAC clones which spanned 500 kb was constructed. All the insert-ends of the BAC clones could be amplified with thermal asymmetric interlaced PCR. Fourteen insert-ends were subcloned. Seven of them were identified as a single or low copy sequences and five were mapped on the expected sites flanking G342 or R1167. The insert fragment isolated from the minimum tile BAC clones of the contig was used to screen a cDNA library and four different positive clones were detected.

Isolation and identification of a non-specific tandemly repeated DNA sequence in Oryza species.

A tandemly repeated DNA sequence (RRS7) was isolated from Oryza alta (CCDD). RRS7-related sequences were also found tandemly arrayed in genomes AA, BB, BBCC, CC, and EE, and a small amount of RRS7-related sequences were detected in genome FF and the Oryza species with unknown genomes. DNA sequence analysis of the 1844-bp insert of RRS7 revealed that it contained six tandemly repeated units, of which five were 155 bp in length and one was 194 bp in length and contained an imperfect internal 39-bp duplication. Southern blot analysis showed that the boundary sequence contained in RRS7 is a single-copy sequence. A 155-bp consensus sequence derived from the six monomeric repeats contained no internal repeat and showed no significant homology to other currently known sequences. The results of Southern blot and sequence analysis revealed that there are at least two subfamilies present in the RRS7 family; these are represented by the DraI site and the MspI site, respectively. Restriction digestion with two pairs of isoschizomers MboI/Sau3A and MspI/HpaII demonstrated that most of the C residues in the GATC sites and the internal C in the CCGG sites of the RRS7 family in O. Alta were methylated. The usefulness of the RRS7 family in determining the evolutionary relationship of the genome DD and other Oryza genomes is discussed.

A receptor kinase-like protein encoded by the rice disease resistance gene, Xa21.

The rice Xa21 gene, which confers resistance to Xanthomonas oryzae pv. oryzae race 6, was isolated by positional cloning. Fifty transgenic rice plants carrying the cloned Xa21 gene display high levels of resistance to the pathogen. The sequence of the predicted protein, which carries both a leucine-rich repeat motif and a serine-threonine kinase-like domain, suggests a role in cell surface recognition of a pathogen ligand and subsequent activation of an intracellular defense response. Characterization of Xa21 should facilitate understanding of plant disease resistance and lead to engineered resistance in rice.