Journal of Genetics and Genomics

Assignment and expression patterns of porcine muscle-specific isoform of phosphoglycerate mutase gene

Haifang Qiu, Shuhong Zhao, Xuewen Xu, Martine Yerle, Bang Liu

2008, 35(5): 257-260. doi: 10.1016/S1673-8527(08)60036-3

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Abstract:
It has been reported that the muscle-specific isoform (type M, PGAM2) of phosphoglycerate mutase (PGAM) is a housekeeping enzyme; it catalyzes the conversion of 3-phosphoglycerate into 2-phosphoglycerate in the glycolysis process to release energy. It is encoded by the Pgam2 gene. In this study, the cDNA of the porcine Pgam2 was cloned. This gene contains an open reading frame of 765 bp encoding a protein of 253 residues, and the predicted protein sequences share high similarity with other mammalians, 96% identity with humans, and 94% identity with mouse and rats. Pgam2 was mapped to SSC18q13-q21 by the RH panel. In this region, there are several QTLs, such as fat ratio, lean percentage, and diameter of muslce fiber, which affect meat production and quality. The reverse transcriptase-polymerase chain reaction revealed that the porcinePgam2 gene was mainly expressed in the muscle tissue (skeletal muscle and cardiac muscle), and was expressed highly at skeletal muscle development stages (embryonic periods: 33, 65, and 90 days post-conception (dpc); postnatal pigs: 4 days and adult). This indicates that the Pgam2 gene plays an important role in muscle growth and development. In addition, it was demonstrated that PGAM2 locates both in cytoplasm and nuclei, and takes part in the glycometabolism process of cytoplasm and nuclei.

Identification of microsatellites in cattle unigenes

Qiuliang Yan, Yinghan Zhang, Hongbin Li, Caihong Wei, Lili Niu, Shan Guan, Shangang Li, Lixin Du

2008, 35(5): 261-266. doi: 10.1016/S1673-8527(08)60037-5

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Abstract:
To identify EST-SSR molecular markers, 41,986 cattle UniGene sequences from NCBI were mined for analyzing SSRs. A total of 1,831 SSRs were identified from 1,666 ESTs, which represented an average density of 19.88 kb per SSR. The frequency of EST-SSRs was 4.0%. The dinucleotide repeat motif was the most abundant SSR, accounting for 54%, followed by 22%, 13%, 7% and 4%, respectively, for tri-, hexa-, penta- and tetra-nucleotide repeats. Depending upon the length of the repeat unit, the length of microsatellites varied from 14 to 86 bp. Among the di- and tri-nucleotide repeats, AC/TG (57%) and AGC (12%) were the most abundant type. Annotation of EST-SSRs was also carried out. Three hundred primer pairs were randomly designed using Prime Premier 5.0 program and Oligo 5.0 for further experimental validation.

Correlation of genomic and expression alterations of AS3 with esophageal squamous cell carcinoma

Yu Zhang, Xiaoping Huang, Jun Qi, Cai Yan, Xin Xu, Yaling Han, Mingrong Wang

2008, 35(5): 267-271. doi: 10.1016/S1673-8527(08)60038-7

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Abstract:
Androgen-induced proliferation shutoff gene AS3, also known as APRIN, is a growth inhibitory gene that is initially implicated in prostate cancer. This gene is required for androgen-dependent growth arrest and is a primary target for 1,25(OH)₂D₃ and androgens. Allelic loss at AS3 locus has been linked to a variety of cancers. However, the correlation of genomic and expression alterations of AS3 with esophageal squamous cell carcinoma (ESCC) is not well established. In this study, the genomic and expression alterations of AS3 in ESCC and their clinical significance are evaluated. Loss of heterozygosity (LOH) analysis using an AS3 intragenic microsatellite marker D13S171 revealed 72% allelic loss at AS3 locus in ESCC, which is significantly correlated with higher pathological grade (P=0.042). RT-PCR examination showed that AS3 mRNA obviously decreased in 44% tumors and its down-regulation was correlated with the sex of patients (P=0.03). Furthermore, the correlation between genomic and expression alterations of AS3 gene was analyzed in 18 ESCC specimens, which indicated that the consistency between allelic loss and decreased mRNA expression of AS3 was relatively poor. The results of this study indicate that the aberrant expression of AS3 may be involved in the tumorigenesis of esophagus and is responsible for the male predominance of ESCC.

Effects of donor cells on in vitro development of cloned bovine embryos

Jing Fu, Pengfei Guan, Leiwen Zhao, Hua Li, Shuzhen Huang, Fanyi Zeng, Yitao Zeng

2008, 35(5): 273-278. doi: 10.1016/S1673-8527(08)60039-9

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Abstract:
The donor cells from different individuals and with different foreign genes introduced were investigated to determine their effects on the efficiency of somatic cell nuclear transfer (SCNT). The bovine ear fibroblast from different individuals was isolated, cultured, and then transfected with foreign genes to establish the stable cell lines, which were used as donor cells for nuclear transfer. The oocytes were obtained through ovum pick up operation. Afterin vitro maturation, the M II phase oocytes were selected as receptors for nuclear transfer. The reconstructed embryos were cultured in vitro and observed at 2 h, 48 h, and 7 days after transfer to assess the rate of fusion using cleaved and blastocyst as the parameters of SCNT efficiency. The donor cells from different individuals (04036, 06081, 06088, and 06129) had no obvious effect on the fusion and cleaved rate, whereas there was significant difference in the blastocyst rate (P<0.05), and the rate was 62.3%, 37.0%, 35.1%, and 15.6%, respectively. There was no significant difference among the rate of fusion, cleaved and blastocyst in donor cells with different foreign genes (P>0.05). It was concluded that the genetic background of the donor cells could affect the efficiency of SCNT, while the introduction of foreign genes into the donor cells had no obvious effect on the efficiency. This study provides useful information for the SCNT and would benefit in promoting the efficiency.

Establishment of paternity testing system using microsatellite markers in Chinese Holstein

Fei Tian, Dongxiao Sun, Yuan Zhang

2008, 35(5): 279-284. doi: 10.1016/S1673-8527(08)60040-5

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Abstract:
To estimate the efficiency of microsatellite markers in paternity testing among Chinese Holstein, 30 microsatellite loci were used to differentiate 330 Chinese Holstein genotypes, according to the calculation of the allele frequency, number of alleles, effective number of alleles, genetic heterozygosity, polymorphic information content (PIC), and the exclusion probability in this cattle population. The results demonstrated that the exclusion probability ranged from 0.620 in locus BM1818 to 0.265 in locus INRA005 with the average of 0.472 and 11 microsatellite markers exceeding 0.5. The combined exclusion probability of nine microsatellite markers was over 0.99. The result showed that paternity testing of Chinese Holstein was basically resolved using the nine microsatellite markers selected.

Phylogenetic analysis of 48 gene families revealing relationships between Hagfishes, Lampreys, and Gnathostomata

Shuiyan Yu, Weiwei Zhang, Ling Li, Huifang Huang, Fei Ma, Qingwei Li

2008, 35(5): 285-290. doi: 10.1016/S1673-8527(08)60041-7

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Abstract:
It has become clear that the extant vertebrates are divided into three major groups, that is, hagfishes, lampreys, and jawed vertebrates. Morphological and molecular studies, however, have resulted in conflicting views with regard to their interrelationships. To clarify the phylogenetic relationships between them, 48 orthologous protein-coding gene families were analyzed. Even as the analysis of 34 nuclear gene families supported the monophyly of cyclostomes, the analysis of 14 mitochondrial gene families suggested a closer relationship between lampreys and gnathostomes compared to hagfishes. Lampreys were sister group of gnathostomes. The results of this study supported the cyclostomes. Choice of outgroup, tree-making methods, and software may affect the phylogenetic prediction, which may have caused much debate over the subject. Development of new methods for tackling such problems is still necessary.

Analysis of genetic relationship in mutant silkworm strains of Bombyx mori using inter simple sequence repeat (ISSR) markers

Dhanikachalam Velu, Kangayam M. Ponnuvel, Murugiah Muthulakshmi, Randhir K. Sinha, Syed M.H. Qadri

2008, 35(5): 291-297. doi: 10.1016/S1673-8527(08)60042-9

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Abstract:
Amplified inter simple sequence repeats (ISSR) markers were used to determine genetic relationships among mutant silkworm strains of Bombyx mori. Fifteen ISSR primers containing simple sequence repeat (SSR) motifs were used in this study. A total of 113 markers were produced among 20 mutant strains, of which 73.45% were found to be polymorphic. In selected mutant genetic stocks, the average number of observed allele was (1.7080 ± 0.4567), effective alleles (1.5194 ± 0.3950) and genetic diversity (Ht) (0.2901 ± 0.0415). The dendrogram produced using the unweighted pair group method with arithmetic means (UPGMA) and cluster analysis made using Nei's genetic distance resulted in the formation of one major group containing 6 groups separated 20 mutant silkworm strains. Therefore, ISSR amplification is a valuable method for determining the genetic variability among mutant silkworm strains. This efficient molecular marker would be useful for characterizing a considerable number of silkworm strains maintained at the germplasm center.

Identification of quantitative trait loci for the dead leaf rate and the seedling dead rate under alkaline stress in rice

Dongling Qi, Guizhen Guo, Myung-chul Lee, Junguo Zhang, Guilan Cao, Sanyuan Zhang, Seok-cheol Suh, Qingyang Zhou, Longzhi Han

2008, 35(5): 299-305. doi: 10.1016/S1673-8527(08)60043-0

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The quantitative trait loci (QTLs) for the dead leaf rate (DLR) and the dead seedling rate (DSR) at the different rice growing periods after transplanting under alkaline stress were identified using an F_2:3 population, which included 200 individuals and lines derived from a cross between two japonica rice cultivars Gaochan 106 and Changbai 9 with microsatellite markers. The DLR detected at 20 days to 62 days after transplanting under alkaline stress showed continuous normal or near normal distributions in F₃ lines, which was the quantitative trait controlled by multiple genes. The DSR showed a continuous distribution with 3 or 4 peaks and was the quantitative trait controlled by main and multiple genes when rice was grown for 62 days after transplanting under alkaline stress. Thirteen QTLs associated with DLR were detected at 20 days to 62 days after transplanting under alkaline stress. Among these,qDLR9-2 located in RM5786-RM160 on chromosome 9 was detected at 34 days, 41 days, 48 days, 55 days, and 62 days, respectively; qDLR4 located in RM3524-RM3866 on chromosome 4 was detected at 34 days, 41 days, and 48 days, respectively; qDLR7-1 located in RM3859-RM320 on chromosome 7 was detected at 20 days and 27 days; and qDLR6-2 in RM1340-RM5957 on chromosome 6 was detected at 55 days and 62 days, respectively. The alleles of both qDLR9-2 and qDLR4 were derived from alkaline sensitive parent “Gaochan106”. The alleles of both qDLR7-1 and qDLR6-2 were from alkaline tolerant parent Changbai 9. These gene actions showed dominance and over dominance primarily. Six QTLs associated with DSR were detected at 62 days after transplanting under alkaline stress. Among these, qDSR6-2 and qDSR8 were located in RM1340-RM5957 on chromosome 6 and in RM3752-RM404 on chromosome 8, respectively, which were associated with DSR and accounted for 20.32% and 18.86% of the observed phenotypic variation, respectively; qDSR11-2 and qDSR11-3 were located in RM536-RM479 and RM2596-RM286 on chromosome 11, respectively, which were associated with DSR explaining 25.85% and 15.41% of the observed phenotypic variation, respectively. The marker flanking distances of these QTLs were quite far except that of qDSR6-2, which should be researched further.

Cloning and expression analysis of GhDET3, a vacuolar H+-ATPase subunit C gene, from cotton

Zhongyi Xiao, Kunling Tan, Mingyu Hu, Peng Liao, Kuijun Chen, Ming Luo

2008, 35(5): 307-312. doi: 10.1016/S1673-8527(08)60044-2

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Abstract:
Vacuolar H⁺-ATPase was regarded as a key enzyme promoting the fiber cell elongation in cotton (Gossypium hirsuturm L.) through regulating turgor-driven pressure involved in polarity expansion of single cell fiber. The DET3, a V-ATPase subunit C, plays an important role in assembling subunits and regulating the enzyme activity, and is involved in Brassinosteroid-induced cell elongation. To analyze the function of GhDET3 on the elongation of cotton fibers, seven candidates of ESTs were screened and contigged for a 5′-upstream sequence, and the 3′-RACE technique was used to clone the 3′-downstream sequence for the full length of GhDET3 gene. The full length of the target clone was 1,340 bp, including a 10 bp 5′-UTR, an ORF of 1,134 bp, and a 196 bp 3′-UTR. This cDNA sequence encoded a polypepide of 377 amino acid residues with a predicted molecular mass of 43 kDa and a basic isoelectric point of 5.58. Furthermore, a length of 3,410 bp sequence from genomic DNA ofGhDET3 was also cloned by PCR. The deduced amino acid sequence had a high homology with DET3 from Arabidopsis, rice, and maize. Quantitative real-time PCR (qRT-PCR) analysis showed that the GhDET3 expression pattern was ubiquitous in all the tissues and organs detected. The result also revealed that the accumulation of GhDET3 mRNA reached the highest profile at the fiber elongation stage in 12 DPA (days post anthesis) fibers, compared with the lowest level at the fiber initiation stage in 0 DPA ovules (with fibers). The transcript accumulation in fibers and ovules shared the similar variation tendency. In addition, in vitro ovule culture experiment demonstrated that exogenous 24-EBL treatment to 4 DPA ovules (with fibers) was capable of increasing the expression level of GhDET3, and the mRNA accumulation of GhDET3 increased in transgenic FBP7::GhDET2 cotton fibers in vivo. These results indicate that GhDET3 gene plays a crucial role in cotton fiber elongation.

Construction of multiform scFv antibodies using linker peptide

Shihua Wang, Cengjie Zheng, Ying Liu, Huirong Zheng, Zonghua Wang

2008, 35(5): 313-316. doi: 10.1016/S1673-8527(08)60045-4

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Abstract:
Multiform single chain variable fragments (scFvs) including different length linker scFvs and bispecific scFv were constructed. The linker lengths of 0, 3, 5, 8, 12, and 15 amino acids between V_H and V_L of antideoxynivalenol (anti-DON) scFv were used to analyze the affinities of scFvs. The affinity constants of these scFvs increased when the linker was lower than 12 amino acids. The affinity constant would not change when the linker was longer than 12 amino acids. Fusion gene of anti-DON scFv and antizearalenone (anti-ZEN) scFv was also constructed through connection by a short peptide linker DNA to express a bispecific scFv. The affinity constants assay showed that the two scFvs of fusion bispecific scFv remained their own affinity compared to their parental scFvs. Competitive direct enzyme linked immunosorbent assay was used to detect DON and ZEN in contaminated wheat (Triticum aestivum L.) samples, and the results indicated that this bispecific scFv was applicable in DON and ZEN detection. This work confirmed that bispecific scFv could be successfully obtained, and might also have an application in diagnosing fungal infection, and breeding transgenic plants.

Instructions for authors

2008, 35(5): 317-320. doi: 10.1016/S1673-8527(08)60046-6

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Abstract:

2008 Vol. 35, No. 5