A20/AN1 zinc-finger domain-containing proteins in plants and animals represent common elements in stress response

A20/AN1 zinc-finger domain-containing proteins are well characterized in animals, and their role in regulating the immune response is established. Recently, such A20/AN1 zinc-finger proteins have been reported from plants. These plant proteins are involved in stress response, but their exact molecular mechanism of action is yet to be deciphered. Sequence information available in public databases has been used to conduct a survey of A20/AN1 zinc-finger proteins across diverse organisms with a special emphasis on plants. Domain analysis provides some interesting insights into their biological function, the most important being that A20/AN1 zinc-finger proteins could represent common elements of stress response in plants and animals.     

Association analysis of ERBB2 amplicon genetic polymorphisms and STARD3 expression with risk of gastric cancer in the Chinese population

The purpose of this study was to investigate whether risk of gastric cancer (GC) was associated with single nucleotide polymorphisms (SNPs) in a gene cluster on the chromosome 17q12-q21 (ERBB2 amplicon) in the Chinese Han population. We detected twenty-six SNPs in this gene cluster containing steroidogenic acute regulatory-related lipid transfer domain containing 3 (STARD3), protein phosphatase 1 regulatory subunit 1B (PPP1R1B/DARPP32), titin-cap (TCAP), per1-like domain containing 1(PERLD1/CAB2), human epidermal growth factor receptor-2 (ERBB2/HER2), zinc-finger protein subfamily 1A 3 (ZNFN1A3/IKZF3) and DNA topoisomerase 2-alpha (TOP2A) genes in 311 patients with GC and in 425 controls by Sequenom. We found no associations between genetic variations and GC risk. However, haplotype analysis implied that the haplotype CCCT of STARD3 (rs9972882, rs881844, rs11869286 and rs1877031) conferred a protective effect on the susceptibility to GC (P = 0.043, odds ratio [OR] = 0.805, 95% confidence intervals [95% CI] = 0.643–0.992). The STARD3 rs1877031 TC genotype endued histogenesis of gastric mucinous adenocarcinoma and signet-ring cell carcinoma (P = 0.021, OR = 2.882, 95% CI = 1.173–7.084). We examined the expression of STARD3 in 243 tumor tissues out of the 311 GC patients and 20 adjacent normal gastric tissues using immumohistochemical (IHC) analysis and tissue microarrays (TMA). The expression of STARD3 was observed in the gastric parietal cells and in gastric tumor tissues and significantly correlated with gender (P = 0.004), alcohol drinking (P < 0.001), tumor location (P = 0.007), histological type (P = 0.005) and differentiation (P = 0.023) in GC. We concluded that the combined effect of haplotype CCCT of STARD3 might affect GC susceptibility. STARD3 expression might be related to the tumorigenesis of GC in the Chinese population.     

Disruption of the Myostatin Gene in Porcine Primary Fibroblasts and Embryos Using Zinc-Finger Nucleases

Myostatin represses muscle growth by negatively regulating the number and size of muscle fibers. Myostatin loss-of-function can result in the double-muscling phenotype and increased muscle mass. Thus, knockout of myostatin gene could improve the quality of meat from mammals. In the present study, zinc finger nucleases, a useful tool for generating gene knockout animals, were designed to target exon 1 of the myostatin gene. The designed ZFNs were introduced into porcine primary fibroblasts and early implantation embryos via electroporation and microinjection, respectively. Mutations around the ZFNs target site were detected in both primary fibroblasts and blastocysts. The proportion of mutant fibroblast cells and blastocyst was 4.81% and 5.31%, respectively. Thus, ZFNs can be used to knockout myostatin in porcine primary fibroblasts and early implantation embryos.     

Genetics and mechanisms of hepatic cystogenesis

Polycystic liver disease (PLD) is a heterogeneous genetic condition. PKD1 and PKD2 germline mutations are found in patients with autosomal dominant polycystic kidney disease (ADPKD). Autosomal dominant polycystic liver disease (ADPLD) is associated with germline mutations in PRKCSH, SEC63, LRP5, and recently ALG8 and SEC61. GANAB mutations are found in both patient groups. Loss of heterozygosity of PLD-genes in cyst epithelium contributes to the development of hepatic cysts. A genetic interaction network is implied in hepatic cystogenesis that connects the endoplasmic glycoprotein control mechanisms and polycystin expression and localization. Wnt signalling could be the major downstream signalling pathway that results in hepatic cyst growth. PLD in ADPLD and ADPKD probably results from changes in one common final pathway that initiates cyst growth. This article is part of a Special Issue entitled: Cholangiocytes in Health and Diseaseedited by Jesus Banales, Marco Marzioni, Nicholas LaRusso and Peter Jansen.     

Salt tolerance in crop plants: new approaches through tissue culture and gene regulation

Recent approaches to study of salinity tolerance in crop plants have ranged from genetic mapping to molecular characterization of gene products induced by salt/drought stress. Transgenic plant design has allowed to test the effects of overexpression of specific prokaryotic or plant genes that are known to be up-regulated by salt/drought stress. This review summarizes current progress in the field in the context of adaptive metabolic and physiological responses to salt stress and their potential role in long term tolerance. Specifically considered are gene activation by salt, in view of proposed avenues for improved salt tolerance and the need to ascertain the additional influences of developmental regulation of such genes. Discussion includes the alternate genetic strategy we have pursued for improving salinity tolerance in alfalfa (Medicago sativa L.) and rice (Oryza sativa L.). This strategy combines single-step selection of salt-tolerant cells in culture, followed by regeneration of salt-tolerant plants and identification of genes important in conferring salt tolerance. We have postulated that activation or improved expression of a subset of genes encoding functions that are particularly vulnerable under conditions of salt-stress could counteract the molecular effects of such stress and could provide incremental improvements in tolerance. We have proceeded to identify the acquired specific changes in gene regulation for our salt-tolerant mutant cells and plants. One particularly interesting and novel gene isolate from the salt-tolerant cells is Alfin1, which encodes a putative zinc-finger regulatory protein, expressed predominantly in roots. We have demonstrated that this protein binds DNA in a sequence specific manner and may be potentially important in gene regulation in roots in response to salt and an important marker for salt tolerance in crop plants.     

PR65A 调控抗病毒蛋白ZAP的活性

锌指结构抗病毒蛋白(ZAP)能够特异性地识别病毒RNA并促进其特异性降解,从而抑制病毒的复制．ZAP不能独立地发挥抗病毒的功能,需要招募细胞内很多因子,包括外切酶复合体(exosome)、RNA解旋酶p72 等．通过蛋白质组学分析方法,我们找到了与ZAP可能存在相互作用的一些蛋白质并证实蛋白磷酸酶2调节亚基A的?琢亚型(PR65A)与ZAP之间存在着直接的相互作用．细胞内PR65A的表达水平,降低削弱ZAP的活性, 表明PR65A是ZAP发挥最佳抗病毒活性所必需的．这丰富了我们对ZAP抗病毒作用机理的认识,同时也为更好地利用其抗病毒活性提供了重要的指导．     

Site-specific integration of retroviral DNA in human cells using fusion proteins consisting of human immunodeficiency virus type 1 integrase and the designed polydactyl zinc-finger protein E2C

During the life cycle of retroviruses, establishment of a productive infection requires stable joining of a DNA copy of the viral RNA genome into host cell chromosomes. Retroviruses are thus promising vectors for the efficient and stable delivery of genes in therapeutic protocols. Integration of retroviral DNA is catalyzed by the viral enzyme integrase (IN), and one salient feature of retroviral DNA integration is its lack of specificity, as many chromosomal sites can serve as targets for integration. Despite the promise for success in the clinic, one major drawback of the retrovirus-based vector is that any unintended insertion events from the therapy can potentially lead to deleterious effects in patients, as demonstrated by the development of malignancies in both animal and human studies. One approach to directing integration into predetermined DNA sites is fusing IN to a sequence-specific DNA-binding protein, which results in a bias of integration near the recognition site of the fusion partner. Encouraging results have been generated in vitro and in vivo using fusion protein constructs of human immunodeficiency virus type 1 IN and E2C, a designed polydactyl zinc-finger protein that specifically recognizes an 18-base pair DNA sequence. This review focuses on the method for preparing infectious virions containing the IN fusion proteins and on the quantitative PCR assays for determining integration site specificity. Efforts to engineer IN to recognize specific target DNA sequences within the genome may lead to development of effective retroviral vectors that can safely deliver gene-based therapeutics in a clinical setting.     

ZFN-induced mutagenesis and gene-targeting in Arabidopsis through Agrobacterium-mediated floral dip transformation

Zinc-finger nucleases (ZFNs) are artificial restriction enzymes, custom designed for induction of double-strand breaks (DSBs) at a specific locus. These DSBs may result in site-specific mutagenesis or homologous recombination at the repair site, depending on the DNA repair pathway that is used. These promising techniques for genome engineering were evaluated in Arabidopsis plants using Agrobacterium -mediated floral dip transformation. A T-DNA containing the target site for a ZFN pair, that was shown to be active in yeast, was integrated in the Arabidopsis genome. Subsequently, the corresponding pair of ZFN genes was stably integrated in the Arabidopsis genome and ZFN activity was determined by PCR and sequence analysis of the target site. Footprints were obtained in up to 2% of the PCR products, consisting of deletions ranging between 1 and 200 bp and insertions ranging between 1 and 14 bp. We did not observe any toxicity from expression of the ZFNs. In order to obtain ZFN-induced gene-targeting (GT), Arabidopsis plants containing the target site and expressing the ZFN pair were transformed with a T-DNA GT construct. Three GT plants were obtained from ∼3000 transformants. Two of these represent heritable true GT events, as determined by PCR, Southern blot analysis and sequencing of the resulting recombined locus. The third plant showed an ectopic GT event. No GT plants were obtained in a comparable number of transformants that did not contain the ZFNs. Our results demonstrate that ZFNs enhance site-specific mutagenesis and gene-targeting of Agrobacterium T-DNA constructs delivered through floral dip transformation.