Journal of Genetics and Genomics

Cloning of Novel Tumor Metastasis-Related Genes from the Highly Metastatic Human Lung Adenocarcinoma Cell Line Anip973

Fangli Liu, Yu Li, Yang Yu, Songbin Fu, Pu Li

2007, 34(3): 189-195. doi: 10.1016/S1673-8527(07)60020-4

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Abstract:
A cDNA library was successfully constructed from Anip973, a human lung adenocarcinoma cell line with high metastatic potential. NIH3T3 cells were stably transfected using this cDNA library and screened for morphological changes in a soft agar assay. Genomic DNA was isolated from putative clones and the integrated sequence was retrieved by PCR and sequencing. Three known genes, ribosomal protein L23, hypothetical protein FLJ22104, and serine protease inhibitor, kazal type 6 and a number of 5′-terminally truncated sequences were identified. Furthermore, cells transfected with ribosomal protein L23 was highly invasive compared with the empty vector as control (P < 0.02). These results indicate that the expression cloning of cDNA libraries in NIH3T3 cells and subsequent screening for loss of contact inhibition in soft agar is a viable tool for identifying tumor-related genes and ribosomal protein L23 gene plays a role in cell movement and metastasis.

Research on the Karyotype and Evolution of Drosophila melanogaster Species Group

Qiuhong Deng, Qingtao Zeng, Yuanhuai Qian, Chunxuan Li, Yong Yang

2007, 34(3): 196-213. doi: 10.1016/S1673-8527(07)60021-6

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Mitotic metaphase chromosomes of 34 species of Drosophila melanogaster species group were examined. Certain new karyotypes were described for the first time, and their evolutionary and interspecific genetic relationships among 8 subgroups of D. melanogaster species group were analyzed systematically. The results were as follows. The basic karyotype of elegans subgroup was type A. The karyotypes of eugracilis subgroup, melanogaster subgroup, and ficusphila subgroup were all type C. The karyotypes of takahashii subgroup and suzukii subgroup were both type C and type D. The montium subgroup had six kinds of karyotypes: types B, C, C', D, D', and E. The ananassae subgroup had three kinds of karyotypes: types F, G, and H. Thus, the melanogaster species group was classified into five pedigrees based on the diversity of these karyotypes: 1) elegans; 2) eugracilis-melanogaster-ficusphila; 3) takkahashii-suzukii; 4) montium; 5) ananassae. The above-mentioned results in karyotypic evolution were consistent with those of DNA sequence analysis reported by Yang except for the elegans subgroup and this subgroup was considered as the ancestral subgroup. Karyotype analysis of the same drosophila from different isofemale lines indicated that the same Drosophila from different places showed karyotypic variation which might be due to different geographical environment and evolutionary degree or interaction between the two factors.

Microsatellite Genotyping for Four Expected Inbred Mouse Strains from KM Mice

Xiaojuan Zhang, Zhaohui Zhu, Zhaofeng Huang, Pingping Tan, Runlin Z. Ma

2007, 34(3): 214-222. doi: 10.1016/S1673-8527(07)60022-8

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Abstract:
Chinese Kun Ming (KM) mouse, an outbreed strain of laboratory animal, has been widely utilized in related pharmaceutical and genetic studies throughout China. However, the value of KM mice to the research community has been severely limited, partially due to the fact that well-characterized inbred strain of KM mice is not available. Several expected inbred strains from KM mice have been bred, but their genetic purity remains uncertain. In this study, four expected inbred strains of KM mice (A1, T2, N2, and N4) were chosen and their inbred degree were compared with two classical inbred mouse lines (BALB/c and C57BL/6) by analyzing the genotypes of about 30 microsatellite markers. In the four strains, A1 and N4 were homozygous at all genotyped loci, but N2 and T2 were only heterozygous at locus D15Mit16. These results indicate that the level of genetic purity/homozygousity of A1, N4, N2, and T2 inbred line is comparable to those of BALB/c and C57BL/6. This study provided the first and solid evidence for genetic purity of four expected inbred strains of KM mice. These 4 inbred mice strains should be well maintained for further characterization and utilization in genetic studies.

Developmental Changes of Col3a1 mRNA Expression in Muscle and Their Association with Intramuscular Collagen in Pigs

Xinjian Bao, Yongqing Zeng, Shudong Wei, Gang Wang, Chanjuan Liu, Yanxiao Sun, Qimei Chen, Hua Li

2007, 34(3): 223-228. doi: 10.1016/S1673-8527(07)60023-X

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Eighty-four castrated boars including Laiwu Black (LW) (weight 30–90 kg, n = 6) and Lulai Black (LL) (weight 40–100 kg, n = 6) were used to study the developmental changes of collagen type ? alpha 1 ( Col3a1) mRNA expression in the muscle and their association with intramuscular collagen (IMC). The muscle total RNA was extracted to determine the abundance of Col3a1 mRNA using relative quantitative RT-PCR with ?-actin mRNA as the internal standard. The results indicated that the developmental patterns of muscle Col3a1 mRNA in LW and LL pigs were similar. The abundance of Col3a1 mRNA increased with body weight, but decreased a little at 70 kg and 80 kg phases for LW and LL, respectively. On the whole, the expression level of Col3a1 mRNA in muscle of LW was higher than that of LL (P < 0.05). Correlation analysis showed that the expression of Col3a1 mRNA in muscle was positively correlated with total and insoluble IMC, but was negatively correlated with IMC solubility for LW pigs (P < 0.01) and LL pigs ( P < 0.05), respectively. These results suggest that the muscle Col3a1 gene expression is affected by body weight and genotype and has important effect on IMC content and characteristics.

A Nucleus-encoded Topological Specificity Factor PpMinE in Physcomitrella patens has Conserved Function Similar to Its Chloroplast-encoded Ancestor

Jiaying Zhu, Weizhong Liu, Weiwei Zhou, Yong Hu, Yikun He

2007, 34(3): 229-238. doi: 10.1016/S1673-8527(07)60024-1

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A nucleus-encoded MinE gene, designated PpMinE, from Physcomitrella patens was identified using RT-PCR. The presence of both N- and C-terminal extensions in PpMinE protein suggested its cyanobacterial origin. The transient expression of PpMinE using green fluorescent protein fusion in tobacco (Nicotiana tabacum L.) indicated that the PpMinE was a chloroplast-targeted protein. Overexpression of PpMinE in Escherichia coli caused division site misplacement and minicell formation, suggesting evolutionary functional conservation of MinE during plant phylogenesis. According to the phylogenetic tree, PpMinE protein has a close relationship with the highland plants, which suggests that the transfer events of MinE gene from plastid to nucleus might have occurred before the origin of the land plants.

Identification of Quantitative Trait Loci for Cold Response of Seedling Vigor Traits in Rice

Longzhi Han, Yongli Qiao, Sanyuan Zhang, Yuanyuan Zhang, Guilan Cao, Jonghwan Kim, Kyuseong Lee, Heejong Koh

2007, 34(3): 239-246. doi: 10.1016/S1673-8527(07)60025-3

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The quantitative trait loci (QTLs) for the seedling vigor traits under 12°C cold water irrigation, such as the seedling height, the seedling fresh weight, the seedling dry weight, and their cold response index, were identified using an F_2–3 population including 200 individuals and lines derived from a cross of indica and japonica “Milyang 23/Jileng 1” with microsatellite markers. All seedling vigor traits exhibited a continuous distribution near normal in F3 lines; these traits were quantitative traits controlled by multiple genes. Twelve QTLs conferring the seedling vigor traits under cold water irrigation were detected on chromosomes 1, 2, 7, 8, and 12, which explained the observed phenotypic variance from 5.2% to 17.9%. Among them, qCSH2 and qCSH12 were located in RM262-RM263 on chromosome 2 and RM270-RM17 on chromosome 12, respectively, which were associated with the seedling height. qSDW12 and qCSDW1 were located in RM19-RM270 on chromosome 12 and RM129-RM9 on chromosome 1, respectively, which were correlated with the seedling dry weight and its cold response index, and the explained 16.6%, 17.9%, 15.9%, and 16.2% of the observed phenotypic variation, respectively. These QTLs alleles were derived from cold-tolerant parent Jileng 1; the gene actions of the two front genes showed their additive effect, and the two genes belind showed dominant and over dominant effects, respectively.

Genome-wide Detection and Analysis of Alternative Splicing for Nucleotide Binding Site-Leucine-Rich Repeats Sequences in Rice

Lianfeng Gu, Rongfa Guo

2007, 34(3): 247-257. doi: 10.1016/S1673-8527(07)60026-5

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Alternative splicing is a major contributor to genomic complexity and proteome diversity, yet the analysis of alternative splicing for the sequence containing nucleotide binding site and leucine-rich repeats (NBS-LRR) domain has not been explored in rice (Oryza sativa L.). Hidden Markov model (HMM) searches were performed for NBS-LRR domain. 875 NBS-LRR-encoding sequences were obtained from the Institute for Genomic Research (TIGR). All of them were used to blast Knowledge-based Oryza Molecular Biological Encyclopaedia (KOME), TIGR rice gene index (TGI), and Universal Protein Resource (UniProt) to obtain homologous full-length cDNAs (FL-cDNAs), tentative consensus sequences, and protein sequences. Alternative splicing events were detected from genomic alignment of FL-cDNAs, tentative consensus sequences, and protein sequences, which provide valuable information on splice variants of genes. These sequences were aligned to the corresponding BAC sequences using the Spidey and Sim4 programs and each of the proteins was aligned by tBLASTn. Of the 875 NBS-LRR sequences, 119 (13.6%) sequences had alternative splicing where multiple FL-cDNAs, TGI sequences and proteins corresponded to the same gene. 71 intron retention events, 20 exon skipping events, 16 alternative termination events, 25 alternative initiation events, 12 alternative 5′ splicing events, and 16 alternative 3′ splicing events were identified. Most of these alternative splices were supported by two or more transcripts. The data sets are available at http://www.bioinfor.org Furthermore, the bioinformatics analysis of splice boundaries showed that exon skipping and intron retention did not exhibit strong consensus. This implies a different regulation mechanism that guides the expression of splice isoforms. This article also presents the analysis of the effects of intron retention on proteins. The C-terminal regions of alternative proteins turned out to be more variable than the N-terminal regions. Finally, tissue distribution and protein localization of alternative splicing were explored. The largest categories of tissue distributions for alternative splicing were shoot and callus. More than one-thirds of protein localization for splice forms was plasma membrane and cytoplasm. All the NBS-LRR proteins for splice forms may have important function in disease resistance and activate downstream signaling pathways.

Rice Repetitive DNA Sequence RRD3: a Plant Promoter and Its Application to RNA Interference

Jian Zhong, Hongbin Wang, Dangquan Zhang, Bing Liu, Jinfa Wang

2007, 34(3): 258-266. doi: 10.1016/S1673-8527(07)60027-7

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Previously, a moderately repetitive DNA sequence (RRD3) was cloned from rice (Oryza sativa L.) by DNA renaturation kinetics. Sequence analysis revealed several conserved promoter motifs, including four TATA-boxes and a CAAT-box, and promoter activity was shown in Escherichia coli and mammalian expression systems. Here, we inserted the RRD3 fragment into the plant promoter-capture vector, pCAMBIA1391Z, and examined whether the RRD3 fragment has promoter activity in plants. Transgenic tobacco and rice calli both showed β-glucuronidase (GUS) activity, indicating that RRD3 can act as a promoter in both monocot and dicot plants. Based on the promoter characteristic of RRD3, we designed a plant universal binary vector, pCRiRRD3, which is suitable for performing researches on plant RNA interference. This vector has two multiple cloning sites to facilitate sense and antisense cloning of the target sequence, separated by an intron fragment of 200 bp. The efficiency of the vector for gene silencing was assayed by histochemical and quantitative fluorometric GUS assays in transgenic tobacco. These research results suggested that this plant RNAi vector pCRiRRD3 can effectively perform gene silencing researches on both monocot and dicot plants.

Cloning and Expression Analysis of PtFATB Gene Encoding the Acyl-acyl Carrier Protein Thioesterase in Populus tomentosa Carr

Zhou Zhou, Deqiang Zhang, Mengzhu Lu

2007, 34(3): 267-274. doi: 10.1016/S1673-8527(07)60028-9

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Acyl-ACP thioesterases (FATs) terminates the fatty acid synthesis and allow the transport of fatty acids out of the plastids, which are the important determinants of cellular metabolism. FATB is a member of FAT enzymes that has been described previously in most of the plants. In silico cloning is a new method that utilizes the bioinformatics on the complete genome and available EST database. In this study, a full-length cDNA clone of PtFATB gene was isolated from Populus tomentosa using this approach. It is 1,450 bp in length and the open reading frame encodes a peptide of 421 amino acids. The predicted amino acid sequence shows significant homology with those from other plant species, which contain typical domains owned by FATB proteins. The transcripts of PtFATB were abundant in leaves, and less in roots detected by using semiquantitative RT-PCR. When the shoots were subjected to the stress treatments (cold, dry, NaCl) and ABA (Abscisic acid), the expression of PtFATB was only slightly reduced under the treatment of low temperature. This suggests that the expression of PtFATB is in a constitutive fashion. This study provides the basis not only for the identification and characterization of this gene but also for the improvement of cold tolerance by controlling the expression of the PtFATB gene in trees in near future.

Analysis of Synonymous Codon Usage in Aeropyrum pernix K1 and Other Crenarchaeota Microorganisms

Peng Jiang, Xiao Sun, Zuhong Lu

2007, 34(3): 275-284. doi: 10.1016/S1673-8527(07)60029-0

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In this study, a comparative analysis of the codon usage bias was performed in Aeropyrum pernix K1 and two other phylogenetically related Crenarchaeota microorganisms (i.e., Pyrobaculum aerophilum str. IM2 and Sulfolobus acidocaldarius DSM 639). The results indicated that the synonymous codon usage in A. pernix K1 was less biased, which was highly correlated with the GC_3S value. The codon usage patterns were phylogenetically conserved among these Crenarchaeota microorganisms. Comparatively, it is the species function rather than the gene function that determines their gene codon usage patterns. A. pernix K1, P. aerophilum str. IM2, and S. acidocaldarius DSM 639 live in differently extreme conditions. It is presumed that the living environment played an important role in determining the codon usage pattern of these microorganisms. Besides, there was no strain-specific codon usage among these microorganisms. The extent of codon bias in A. pernix K1 and S. acidocaldarius DSM 639 were highly correlated with the gene expression level, but no such association was detected in P. aerophilum str. IM2 genomes.

2007 Vol. 34, No. 3