Journal of Genetics and Genomics

Patterning mechanisms controlling digit development

Jianxin Hu, Lin He

2008, 35(9): 517-524. doi: 10.1016/S1673-8527(08)60071-5

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Abstract:
Vertebrate digits are essential structures for movement, feeding and communication. Specialized regions of the developing limb bud including the zone of polarizing activity (ZPA), the apical ectodermal ridge (AER), and the non-ridge ectoderm regulate the patterning of digits. Although a series of signaling molecules have been characterized as patterning signals from the organizing centers, the delicate cellular and molecular mechanisms that interpret how these patterning signals control the detailed digit anatomy remain unclear. Recent studies from model organisms and human hand malformations provide new insights into the mechanisms regulating this process. Here, we review the current understanding of the genetic networks governing digit morphogenesis.

Collagen1α1 promoter drives the expression of Cre recombinase in osteoblasts of transgenic mice

Lagabaiyila Zha, Ning Hou, Jian Wang, Guan Yang, Yuanrong Gao, Lin Chen, Xiao Yang

2008, 35(9): 525-530. doi: 10.1016/S1673-8527(08)60072-7

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Abstract:
Osteoblasts participate in bone formation, bone mineralization, osteoclast differentiation and many pathological processes. To study the function of genes in osteoblasts using Cre-LoxP system, we generated a mouse line expressing the Cre recombinase under the control of the rat Collagen1α1 (Col1α1) promoter (Col1α1-Cre). Two founders were identified by genomic PCR from 16 offsprings, and the integration efficiency is 12.5%. In order to determine the tissue distribution and the activity of Cre recombinase in the transgenic mice, the Col1α1-Cre transgenic mice were bred with the ROSA26 reporter strain and a mouse strain that carries Smad4 conditional alleles (). Multiple tissue PCR of mice revealed the restricted Cre activity in bone tissues containing osteoblasts and tendon. LacZ staining in the Col1α1-Cre;ROSA26 double transgenic mice revealed that the Cre recombinase began to express in the osteoblasts of calvaria at E14.5. Cre activity was observed in the osteoblasts and osteocytes of P10 double transgenic mice. All these data indicated that the Col1α1-Cre transgenic mice could serve as a valuable tool for osteoblast lineage analysis and conditional gene knockout in osteoblasts.

Calcineurin B-like interacting protein kinase OsCIPK23 functions in pollination and drought stress responses in rice (Oryza sativa L.)

Wenqiang Yang, Zhaosheng Kong, Edith Omo-Ikerodah, Wenying Xu, Qun Li, Yongbiao Xue

2008, 35(9): 531-543. doi: 10.1016/S1673-8527(08)60073-9

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Abstract:
Drought is very harmful to grain yield due to its adverse effect on reproduction, especially on pollination process in rice. However, the molecular basis of such an effect still remains largely unknown. Here, we report the role of a member of CBL (Calcineurin B-Like) Interacting Protein Kinase (CIPK) family, OsCIPK23, in pollination and stress responses in rice. Molecular analyses revealed that it is mainly expressed in pistil and anther but up-regulated by pollination, as well as by treatments of various abiotic stresses and phytohormones. RNA interference-mediated suppression ofOsCIPK23 expression significantly reduced seed set and conferred a hypersensitive response to drought stress, indicating its possible roles in pollination and drought stress. In consistent, overexpression of OsCIPK23 induced the expression of several drought tolerance related genes. Taken together, these results indicate that OsCIPK23 is a multistress induced gene and likely mediates a signaling pathway commonly shared by both pollination and drought stress responses in rice.

DNA sequencing by synthesis with degenerate primers

Chao Tang, Xiaolong Shi, Xiujie Li, Zuhong Lu

2008, 35(9): 545-551. doi: 10.1016/S1673-8527(08)60074-0

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Abstract:
The degenerate primer-based sequencing was developed by a synthesis method (DP-SBS) for high-throughput DNA sequencing, in which a set of degenerate primers are hybridized on the arrayed DNA templates and extended by DNA polymerase on microarrays. In this method, a different set of degenerate primers containing a given number (n) of degenerate nucleotides at the 3′-ends were annealed to the sequenced templates that were immobilized on the solid surface. The nucleotides (n+1) on the template sequences were determined by detecting the incorporation of fluorescent labeled nucleotides. The fluorescent labeled nucleotide was incorporated into the primer in a base-specific manner after the enzymatic primer extension reactions and nine-base length were read out accurately. The main advantage of the DP-SBS is that the method only uses very conventional biochemical reagents and avoids the complicated special chemical reagents for removing the labeled nucleotides and reactivating the primer for further extension. From the present study, it is found that the DP-SBS method is reliable, simple, and cost-effective for laboratory-sequencing a large amount of short DNA fragments.

Novel ACTG1 mutation causing autosomal dominant non-syndromic hearing impairment in a Chinese family

Ping Liu, Hu Li, Xiang Ren, Haiyan Mao, Qihui Zhu, Zhengfeng Zhu, Rong Yang, Wenlin Yuan, Jingyu Liu, Qing Wang, Mugen Liu

2008, 35(9): 553-558. doi: 10.1016/S1673-8527(08)60075-2

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Abstract:
The γ-actin (ACTG1) gene is a cytoplasmic nonmuscle actin gene, which encodes a major cytoskeletal protein in the sensory hair cells of the cochlea. Mutations in ACTG1 were found to cause autosomal dominant, progressive, sensorineural hearing loss linked to the DFNA 20/26 locus on chromosome 17q25.3 in European and American families, respectively. In this study, a novel missense mutation (c.364A>G; p.I122V) co-segregated with the affected individuals in the family and did not exist in the unaffected family members and 150 unrelated normal controls. The alteration of residue Ile122 was predicted to damage its interaction with actin-binding proteins, which may cause disruption of hair cell organization and function. These findings strongly suggested that the I122V mutation in ACTG1 caused autosomal dominant non-syndromic hearing impairment in a Chinese family and expanded the spectrum ofACTG1 mutations causing hearing loss.

Aberrant DNA methylation in 5′ regions of DNA methyltransferase genes in aborted bovine clones

Jinghe Liu, Xingwei Liang, Jiaqiao Zhu, Liang Wei, Yi Hou, Da-Yuan Chen, Qing-Yuan Sun

2008, 35(9): 559-568. doi: 10.1016/S1673-8527(08)60076-4

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Abstract:
High rate of abortion and developmental abnormalities is thought to be closely associated with inefficient epigenetic reprogramming of the transplanted nuclei during bovine cloning. It is known that one of the important mechanisms for epigenetic reprogramming is DNA methylation. DNA methylation is established and maintained by DNA methyltransferases (DNMTs), therefore, it is postulated that the inefficient epigenetic reprogramming of transplanted nuclei may be due to abnormal expression of DNMTs. Since DNA methylation can strongly inhibit gene expression, aberrant DNA methylation of DNMT genes may disturb gene expression. But presently, it is not clear whether the methylation abnormality of DNMT genes is related to developmental failure of somatic cell nuclear transfer embryos. In our study, we analyzed methylation patterns of the 5′ regions of four DNMT genes includingDnmt3a, Dnmt3b, Dnmt1 and Dnmt2 in four aborted bovine clones. Using bisulfite sequencing method, we found that 3 out of 4 aborted bovine clones (AF1, AF2 and AF3) showed either hypermethylation or hypomethylation in the 5′ regions of Dnmt3a and Dnmt3b, indicating that Dnmt3a and Dnmt3b genes are not properly reprogrammed. However, the individual AF4 exhibited similar methylation level and pattern to age-matched in vitro fertilized (IVF) fetuses. Besides, we found that the 5′ regions of Dnmt1 and Dnmt2 were nearly completely unmethylated in all normal adults, IVF fetuses, sperm and aborted clones. Together, our results suggest that the aberrant methylation of Dnmt3a and Dnmt3b 5′ regions is probably associated with the high abortion of bovine clones.

Identification of quantitative trait loci for four morphologic traits under water stress in rice (Oryza sativa L.)

Bing Yue, Weiya Xue, Lijun Luo, Yongzhong Xing

2008, 35(9): 569-575. doi: 10.1016/S1673-8527(08)60077-6

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Abstract:
Late season drought coinciding with the rice booting to heading stage affects the development of plant height, panicle exsertion, and flag leaf size, and causes significant yield loss. In this study, a recombinant inbred line population derived from a cross between paddy and upland cultivars was used for data collection of the morphologic traits under well water and drought stress conditions. Drought stress was applied at the stage of panicle initiation in the field in 2002 and at the booting stage in PVC pipes in 2003. The data from stress conditions and their ratios (trait measured under stress condition/trait measured under well water condition) or differences (trait measured under stress condition minus trait measured under well water condition) were used for QTL analysis. Totally, 17 and 36 QTLs for these traits were identified in 2002 and 2003, respectively, which explained a range of 2.58%–29.82% of the phenotypic variation. Among them, six QTLs were commonly identified in the two years, suggesting that the drought stress in the two years was different. The genetic basis of these traits will provide useful information for improving rice late season drought resistance, and their application as indirect indices in rice late season drought resistance screening was also discussed.

2008 Vol. 35, No. 9