[Denaturing high-performance liquid chromatography coupled with multiplex PCR for rapid detection of large duplications or deletions in patients with Duchenne muscular dystrophy and spinal muscular atrophy].

OBJECTIVE: To assess the value of multiplex PCR-denaturing high-performance liquid chromatography (PCR-DHPLC) method for screening large duplications or deletions in patients with Duchenne muscular dystrophy (DMD) and spinal muscular atrophy (SMA). METHODS: DNA was extracted from peripheral venous blood samples from 35 DMD and 6 SMA patients. Large duplications or deletions were screened with multiplex PCR coupled with DHPLC method. The results were validated with testing of positive and negative controls. RESULTS: Known duplications or deletions in all controls were reliably detected with multiple PCR coupled with DHPLC. Large duplications or deletions were found in 71.4% of 35 DMD patients, which included 5 large duplications and 20 large deletions. For SMA patients, deletions of SMN1 exon 7 were detected in 16 samples. CONCLUSION: Multiplex PCR coupled with DHPLC method is an effective and reliable method for detecting large genomic duplications or deletions in patients with DMD or SMA.

The complete mitochondrial genome of Platysmacheilus nudiventris (Teleostei, Cyprinidae, Gobioninae).

The complete mitochondrial genome of Platysmacheilus nudiventris (Teleostei, Cyprinidae, Gobioninae), an endemic Chinese cyprinid fish, was first determined by DNA sequencing based on the PCR fragments. The total length of the mitochondrial genome is 16,603 bp, including 22 transfer RNA genes, 2 ribosomal RNA genes, 13 protein-coding genes and a non-coding control region (D-loop). The overall-based composition is 30.2% A, 26.9% T, 16.7% G and 26.1% C, with high A+T content (57.1%). The results will provide useful data for further studies on species identification, evolutionary and population genetic studies of P. nudiventris and its related species.

The complete mitochondrial genome of Xenophysogobio nudicorpa (Cypriniformes; Cyprinidae).

In this study, the complete mitochondrial DNA genome sequence of Xenophysogobio nudicorpa was first determined by DNA sequencing based on the PCR fragments. The complete mitochondrial DNA (mtDNA) genome sequence of X. nudicorpa is a circular molecule of 16,616 bp in length. It consists of 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and a control region (D-loop). The gene nucleotide composition of X. nudicorpa is 30.5% A, 26.0% C, 16.8% G, and 26.7% T, with a relatively high A+T content (57.2%). The results could provide useful data for studying genetic diversity and phylogenetics in X. nudicorpa and related species.

The complete mitochondrial genome of Sinibrama taeniatus (Cyprinidae: Cultrinae).

In this study, the complete mitochondrial DNA genome sequence of Sinibrama taeniatus was first determined by DNA sequencing based on the PCR fragments. The complete mitochondrial DNA (mtDNA) genome sequence of S. taeniatus was a circular molecule of 16,623 bp in length. It consisted of 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and a control region (D-loop). The gene nucleotide composition of S. taeniatus was 31.3% A, 26.8% C, 16.1% G, and 25.8% T, with a relatively high A + T content (57.1%). The results could provide useful data for studying genetic diversity and phylogenetics in S. taeniatus and related species.

The complete mitochondrial genome of Pseudobagrus emarginatus (Siluriformes: Bagridae).

In this study, the total mitochondrial genome sequence of Pseudobagrus emarginatus (Siluriformes: Bagridae) was firstly sequenced and determined. The complete mtDNA genome sequence of P. emarginatus is 16,534 bp in length. It consists of 22 transfer RNA genes, 2 ribosomal RNA genes, 13 protein-coding genes, and a non-coding control region (D-loop). The overall-based composition was 31.5% A, 26.8% T, 14.9% G and 26.8% C, with a high A+T content (58.3%), which was nearly the same as other reported catfishes. These results will provide useful data to the natural resources conservation and systematics analysis of P. emarginatus and its related species in future.

The complete mitochondrial genome of Rhinogobio typus (Teleostei, Cyprinidae, Gobioninae).

In the present study, the complete mitochondrial genome sequence of Rhinogobio typus (Teleostei, Cyprinidae, Gobioninae) was obtained using a PCR-based method. The total length of the mitochondrial genome is 16,608 bp, including 22 transfer RNA genes, 2 ribosomal RNA genes, 13 protein-coding genes, and a non-coding control region (D-loop), which is similar to that observed in many other reported fishes. The overall-based composition is 31.3% A, 26.6% T, 15.9% G and 26.2% C, with high A + T content (57.9%). These results will provide a useful tool for species identification, evolutionary and population genetic studies of R. typus and its related species.

Complete mitochondrial genome of Pareuchiloglanis sinensis (Siluriformes: Sisoridae).

The complete mitochondrial genome of Pareuchiloglanis sinensis, an endemic Chinese sisorid fish, was determined by DNA sequencing based on the PCR fragments. The complete mtDNA genome sequence of P. sinensis is 16,593 bp in length, with a gene content of 13 protein-coding, 2 ribosomal RNA, and 22 transfer RNA genes, and a typical gene arrangement identical to many other reported fishes. The overall base composition of the heavy strand was 30.6% A, 24.2% T, 16.0% G and 29.2% C, with a relatively high A + T content (54.8%). This will provide a useful tool for understanding the genetic diversity, population structure and conservation status of P. sinensis in future.

The complete mitochondrial genome of Glyptothorax sinense (Siluriformes, Sisoridae).

Glyptothorax sinense (Siluriformes, Sisoridae), is a kind of small-sized freshwater fish which mainly distributes in the middle and upper reaches of the Yangtze River in China. In this study, the complete mitochondrial genome of G. sinense was first determined using a PCR-based method. The complete mtDNA sequence is 16,531 bp in length, including 22 transfer RNA genes, 2 ribosomal RNA genes, 13 protein-coding genes, and a non-coding control region (D-loop). The overall-based composition was 31.61% A, 26.66% T, 15.38% G and 26.34% C, with a relatively high A + T content (58.27%). This will provide a useful tool for evolutionary and population genetic studies of G. sinense.

The complete mitochondrial genome of Lepturichthys fimbriata (Teleostei, Balitoridae, Balitorinae).

Lepturichthys fimbriata is an endemic and an important commercial fish which distributes in the upper stream of the Yangtze River. In this study, the complete mitogenome sequence of L. fimbriata has been first sequenced by DNA sequencing based on the PCR fragments. The mitogenome, consisting of 16,567 base pairs (bp), includes 13 protein coding genes, 22 transfer RNAs, 2 ribosomal RNA genes and a non-coding control region (CR). The overall base composition of L. fimbriata is 30.4% A, 28.7% C, 16.5% G, and 24.4% T, with a relatively a slight AT bias of 54.7%. CR of 903 bp length is located between tRNA(Pro) and tRNA(Phe). The complete mitochondrial genome sequence would be useful for further studies on conservation genetics and resource management in L. fimbriata.

The complete mitochondrial genome of Jinshaia sinensis (Teleostei, Balitoridae, Balitorinae).

Jinshaia sinensis is an endemic and typical fish which is successfully adaptive to mountain torrents in the upper stream of the Yangtze River and its tributaries. In this study, the complete mitogenome sequence of J. sinensis has been first sequenced by DNA sequencing based on the PCR fragments. The mitogenome, consisting of 16,567 base pairs (bp), had typical vertebrate mitochondrial gene arrangement, including 13 protein coding genes, 22 transfer RNAs, 2 ribosomal RNA genes and a noncoding control region (CR). The overall base composition of J. sinensis is 30.37% A, 28.82% C, 16.59% G, and 24.22% T, with a relatively a slight AT bias of 54.59%. CR of 903 bp length is located between tRNA(Pro) and tRNA(Phe). The complete mitochondrial genome may provide fundamental informative data not only for unravel the population structure and differentiation, but also for further conservation genetics studies on this balitorid species.

[Analysis of suppressive role of RASSF1A gene at 3p21.3 in lung cancer cell line A549].

OBJECTIVE: Loss of heterozygosity (LOH) at 3p21.3 is a common and early event in the development of lung cancer, implying the presence of tumor suppressor genes (TSGs) that may be involved in the pathogenesis of lung cancer. RASSF1A gene, located at this region, containing the Ras-associated domain, has been considered as a candidate tumor suppressor gene because of high frequency of loss of expression and aberrant promoter hypermethylation in most NSCLCs and SCLCs. This study was designed to transfer the expression vector containing RASSF1A to NSCLC cell line A549 to analyze the effect of this gene's exogenous expression. METHODS: Transfer the pcDNA3.1 and pcDNA3.1-RASSF1A into A549 cells by lipofectamine 2000 respectively, the apoptosis and the changes of cell cycle of transient-transfected cells were analyses by flow cytometry (FCM), cellular proliferation of the stable-transfected was examined by MTT assay, cell growth curve and colony formation assay. Inhibition of lung metastasis in nude mice of RASSF1A exogenous expression was also investigated. RESULTS: The progression of the cell cycle was arrested in G1-phase remarkably induced by RASSF1A (P < 0.001); the cell viability, growing speed and colony formation ability were decreased obviously by exogenous expression of RASSF1A (P < 0.01). The lung metastasis in nude mouse was also significantly inhibited (P < 0.01). CONCLUSION: The experimental results show that RASSF1A plays an important roles in origination, progression and metastasis of lung cancer.

[Relationship between overexpression of the RIalpha subunit of the cAMP-dependent protein kinase and clinicopathological features of lung cancer].

OBJECTIVE: To explore the relationship between the overexpression of PKA RIalpha mRNA and cliniopathological parameters in lung cancer. METHODS: RT-PCR was used to detect the expression of PKA RIalpha mRNA in 54 cases with human lung cancer and matched normal tissues. RESULTS: (1) The expression of PKA RIalpha mRNA was significantly higher in cancer tissue (66.7%) than in normal tissues (20.4%) (P < 0.01). (2) The expression was significantly correlated with TNM stage (P < 0.01), being increased with TNM stage. (3) The expression was significantly higher in patients with positive lymph nodes than in those with negative lymph nodes (P < 0.01). (4) There were no significant associations of PKA RIalpha mRNA expression with histological type, differentiation grade or size of the tumor. CONCLUSION: This study indicates that the overexpression of PKA RIalpha mRNA may play an important role in the progression, metastasis and prognosis of lung cancer.

[Expression of different RASSF1 transcripts and its clinical significance in lung carcinoma].

OBJECTIVE: To evaluate the expression of three different RASSF1 transcripts and its clinical significance in lung carcinomas. METHODS: The mRNA expression of RASSF1A, RASSF1B and RASSF1C was detected by RT-PCR in 51 human lung cancer tissues and 51 matched normal tissues. RESULTS: 1. The mRNA expression of three RASSF1 transcripts was detectable in all non-cancer tissues. However, high rate of expression loss of RASSF1A and RASSF1B existed in lung cancer tissues, which was 53.2% (2851) and 37.3% (19/51), respectively. RASSF1C was expressed in all of the tumor tissues. 2. Loss or abnormal down-regulation of RASSF1A was positively related with lymph node metastasis and TNM stage (P < 0.05) and 3. RASSF1B and RASSF1C mRNA expression was not correlated with TNM stage, histological type, differentiation grade or smoking index. CONCLUSION: There is a significant expression difference among the three RASSF1 transcripts in lung carcinoma. RASSF1A, closely associated with lymph metastasis and TNM stage of lung carcinoma, should be a new tumor suppressor gene.

[Transcriptional inactivation of RASSF1A in lung cancer and its clinical significance].

OBJECTIVE: To explore the relationship between the transcriptional expression of RASSF1A (Ras association domain family 1A gene) and oncogenesis and development of lung cancer. METHOD: RT-PCR was used to detect the expression of RASSF1A mRNA in 47 human lung cancer tissues and matched 47 non-cancer tissues. RESULTS: (1). The RASSF1A mRNA was identified in all non-cancer tissues but not found in 53.2% carcinoma tissues. (2). The rate of loss of RASSF1A mRNA was significantly higher in the patients with positive lymph node metastasis (70.4%) than in those without lymph node metastasis (30.0%) (P < 0.01). (3). Much more frequent in advanced tumor tissues, loss of RASSF1A mRNA was correlated with TNM stage (P < 0.05). (4). No significant association of abnormal RASSF1A expression was identified with histological type, differentiation grade of tumors or age, sex, and smoking index of the patients (all P > 0.05). CONCLUSION: RASSF1A is one of the new candidates of tumor suppressor genes. Loss or abnormal down-regulation of RASSF1A mRNA is a frequent event in lung carcinogenesis, which may play an important role in the malignant progression and prognosis of lung cancer.

[Expression of lysyl oxidase gene in upper digestive tract carcinomas and its clinical significance].

BACKGROUND & OBJECTIVE: Lysyl oxidase(LO), one of newly discovered candidate of tumor suppressor genes, was reported that its expression was closely associated with recurrence and metastasis of breast and prostate carcinomas. However, till now there was no report evaluating the relationship between this gene expression and relapse or metastasis of upper digestive tract carcinomas. This study was designed to explore the relationship between the expression of LO gene and recurrence as well as metastasis of malignant upper digestive tract tumors such as esophageal, cardiac, and gastric cancers. METHOD: LO expression in esophageal, cardiac, and gastric cancer was determined by reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: RT-PCR amplification demonstrated that the expression rate was 42.1% (16/38), 42.9% 9(9/21), and 28.0% (7/25) in esophageal, cardiac, and gastric cancers, respectively. Furthermore, the expression rate of LO mRNA was lower in the tumor tissues with lymph nodes metastasis. And this was also observed in tumor with deep muscular layer infiltration(P < 0.05). In contrast, the reduced expression of the LO gene had no relation to histological classification, invasion pattern, tumor size, and number of metastasis lymph nodes(P > 0.05). CONCLUSIONS: These findings suggest that LO gene may play an important role in metastasis of esophageal, cardiac, and gastric carcinomas.