转基因植物中外源基因的整合特性及其研究策略

综述了外源基因在宿主中的整合特性及结构变化的多样性。将外源基因在宿主中的整合方式分为3种：单拷贝单位点整合、串联多联体单位点整合及单拷贝或多拷贝在多位点整合。分析了不同整合方式对表达的影响。详细讨论了植物转基因的整合机制,阐述了以基因组的双链断裂修复为基础的整合模型。最后介绍了国内外的研究者对不同的整合方式所采取的研究策略。     

转基因植物中外源基因的整合特性及其研究策略

综述了外源基因在宿主中的整合特性及结构变化的多样性.将外源基因在宿主中的整合方式分为3种:单拷贝单位点整合、串联多联体单位点整合及单拷贝或多拷贝在多位点整合.分析了不同整合方式对表达的影响.详细讨论了植物转基因的整合机制,阐述了以基因组的双链断裂修复为基础的整合模型.最后介绍了国内外的研究者对不同的整合方式所采取的研究策略.     

Selection,Morphological Integration,and Strepsirrhine Locomotor Adaptations

Clades with taxa that have multiple locomotor adaptations represent a direct way to test the relationship between adaptation and integration. If integration is influenced by functional requirements, integration should be most apparent where selection is strongest and less evident where selection has been relaxed. If integration is primarily regulated by genetic constraints, integration should be present irrespective of selection pressures. Here we use patterns of integration in the strepsirrhine fore- and hind limbs as a test case. Strepsirrhine locomotion is relatively well-studied, and there are multiple clades that share different locomotor modes. We found that quadrupeds have greater limb integration than vertical leapers. These results suggest that variation can be expressed if selection for integration is relaxed. However, an unexpected pattern was revealed, in which there appears to be some broader regulatory mechanism controlling overall limb integration. Our tests identified a strong correlation between integration of the forelimb and integration of the hind limb. This broader mechanism may be evidence of the primitive genetic control of limb integration.     

Gammaretroviral integration into nucleosomal target DNA in vivo

Some of the earliest studies of retroviral integration targeting reported that sites of gammaretroviral DNA integration were positively correlated with DNase I-hypersensitive sites in chromatin. This led to the suggestion that open chromatin was favorable for integration. More recent deep sequencing experiments confirmed that gammaretroviral integration sites and DNase I cleavage sites are associated in genome-wide surveys. Paradoxically, in vitro studies of integration show that nucleosomal DNA is actually favored over naked DNA, raising the question of whether integration target DNA in chromosomes is wrapped in nucleosomes or nucleosome free. In this study we examined gammaretroviral integration by infecting primary human CD4(+) T lymphocytes with a murine leukemia virus (MLV)-based retroviral vector or xenotropic murine leukemia virus-related virus (XMRV), and isolated 32,585 unique integration sites using ligation-mediated PCR and 454 pyrosequencing. CD4(+) T lymphocytes were chosen for study because of the particularly dense genome-wide mapping of chromatin features available for comparison. Analysis relative to predicted nucleosome positions showed that gammaretroviruses direct integration into outward-facing major grooves on nucleosome-wrapped DNA, similar to the integration pattern of HIV. Also, a suite of histone modifications correlated with gene activity are positively associated with integration by both MLV and XMRV. Thus, we conclude that favored integration near DNase I-hypersensitive sites does not imply that integration takes place exclusively in nucleosome-free regions.     

The adaptive value of phenotypic floral integration

Floral integration has been deemed an adaptation to increase the benefits of animal pollination, yet no attempts have been made to estimate its adaptive value under natural conditions. Here, the variation in the magnitude and pattern of phenotypic floral integration and the variance-covariance structure of floral traits in four species of Rosaceae were examined. The intensity of natural selection acting on floral phenotypic integration was also estimated and the available evidence regarding the magnitude of floral integration reviewed. The species studied had similar degrees of floral integration, although significant differences were observed in their variance-covariance structure. Selection acted on subsets of floral traits (i.e. selection on intrafloral integration) rather than on the integration of the whole flower. Average integration was 20% and similar to the estimated mean value of flowering plants. The review indicated that flowering plants present lower integration than expected by chance. Numerical simulations suggest that this pattern may result from selection favouring intrafloral integration. Phenotypic integration at the flower level seems to have a low adaptive value among the species surveyed. Moreover, it is proposed that pollinator-mediated selection promotes the evolution of intrafloral integration.     

ADAPTIVE RADIATIONS,ECOLOGICAL SPECIALIZATION,AND THE EVOLUTIONARY INTEGRATION OF COMPLEX MORPHOLOGICAL STRUCTURES

The evolutionary integration of complex morphological structures is a macroevolutionary pattern in which morphogenetic components evolve in a coordinated fashion, which can result from the interplay among processes of developmental, genetic integration, and different types of selection. We tested hypotheses of ecological versus developmental factors underlying patterns of within‐species and evolutionary integration in the mandible of phyllostomid bats, during the most impressive ecological and morphological radiation among mammals. Shape variation of mandibular morphogenetic components was associated with diet, and the transition of integration patterns from developmental to within‐species to evolutionary was examined. Within‐species (as a proxy to genetic) integration in different lineages resembled developmental integration regardless of diet specialization, however, evolutionary integration patterns reflected selection in different mandibular components. For dietary specializations requiring extensive functional changes in mastication patterns or biting, such as frugivores and sanguivores, the evolutionary integration pattern was not associated with expected within‐species or developmental integration. On the other hand, specializations with lower mastication demands or without major functional reorganization (such as nectarivores and carnivores), presented evolutionary integration patterns similar to the expected developmental pattern. These results show that evolutionary integration patterns are largely a result of independent selection on specific components regardless of developmental modules.     

Impact of non-homologous end-joining deficiency on random and targeted DNA integration: implications for gene targeting

In higher animal cells, the principal limitation of gene-targeting technology is the extremely low efficiency of targeted integration, which occurs three to four orders of magnitude less frequently than random integration. Assuming that random integration mechanistically involves non-homologous end-joining (NHEJ), inactivation of this pathway should reduce random integration and may enhance gene targeting. To test this possibility, we examined the frequencies of random and targeted integration in NHEJ-deficient chicken DT40 and human Nalm-6 cell lines. As expected, loss of NHEJ resulted in drastically reduced random integration in DT40 cells. Unexpectedly, however, this was not the case for Nalm-6 cells, indicating that NHEJ is not the sole mechanism of random integration. Nevertheless, we present evidence that NHEJ inactivation can lead to enhanced gene targeting through a reduction of random integration and/or an increase in targeted integration by homologous recombination. Most intriguingly, our results show that, in the absence of functional NHEJ, random integration of targeting vectors occurs more frequently than non-targeting vectors (harboring no or little homology to the host genome), implying that suppression of NHEJ-independent random integration events is needed to greatly enhance gene targeting in animal cells.     

Analysis of integration activity of human immunodeficiency virus type-1 integrase.

The integration activity of human immunodeficiency virus type-1 (HIV-1) integrase was characterized in vitro by using pre-processed oligonucleotide substrates. The highest level of integration activity was found at pH 6.5 to 7.0, while the endonucleolytic activity was highest at pH 7.4 to 8.0. Although the endonucleolytic and integration reactions are consecutive in retroviral integration, our result indicates that the optimal conditions of the two reactions are quite different. In addition, it is suggested that the endonucleolytic and integration steps can be separated by control of the cellular physiological state in retroviral therapy. Strong integration was detected in the presence of 0.5-10 mM Mn2+ ion, but weak integration at around 10 mM Mg2+ ion. This observation explains that the Mn2+ ion is preferred to the Mg2+ ion as a cofactor in the integration reaction. Although there was no sequence-specificity in the integration site of the target DNA, integration was found to frequently occur at particular regions of the target DNA. Furthermore, the mutant integrases such as Asp116, Ser147, and Glu152, which had been reported previously, were shown to lose integration activity completely, indicating that these residues are critically involved in catalytic action.     

Sequence requirements for integration of Moloney murine leukemia virus DNA in vitro.

Normal replication of Moloney murine leukemia virus (MoMLV) requires the integration of a DNA copy of the viral RNA genome into a chromosome of the host. In this work, we characterize the DNA sequences at the ends of the linear proviral precursor that are required for integration in the presence of MoMLV integration protein in vitro. We found that nine bases of MoMLV DNA at each end of a linear model substrate were sufficient for near-maximal levels of integration and that four bases of MoMLV DNA at each end were sufficient for low levels of correct integration. We also found that a 3'-terminal A residue was preferred for integration. We infer from the limited DNA sequence requirements for integration that factors in addition to DNA sequence direct integration protein to act at the ends of the viral DNA.     

Integration specificity of the hobo element of Drosophila melanogaster is dependent on sequences flanking the integration site

We analyzed the integration specificity of the hobo transposable element of Drosophila melanogaster. Our results indicate that hobo is similar to other transposable elements in that it can integrate into a large number of sites, but that some sites are preferred over others, with a few sites acting as integration hot spots. A comparison of DNA sequences from 112 hobo integration sites identified a consensus sequence of NTNNNNAC, but this consensus was insufficient to account for the observed integration specificity. To begin to define the parameters affecting hobo integration preferences, we analyzed sequences flanking a donor hobo element, as well as sequences flanking a hobo integration hot spot for their relative influence on hobo integration specificity. We demonstrate experimentally that sequences flanking a hobo donor element do not influence subsequent integration site preference, whereas, sequences contained within 31 base pairs flanking an integration hot spot have a significant effect on the frequency of integration into that site. However, sequence analysis of the DNA flanking several hot spots failed to identify any common sequence motif shared by these sites. This lack of primary sequence information suggests that higher order DNA structural characteristics of the DNA and/or chromatin may influence integration site selection by the hobo element. This revised version was published online in July 2006 with corrections to the Cover Date.     

Site-specific gene integration technologies for crop improvement

Targeted integration of foreign genes into plant genomes is a much sought-after technology for engineering precise integration structures. Homologous recombination-mediated targeted integration into native genomic sites remained somewhat elusive until made possible by zinc finger nuclease-mediated double-stranded breaks. In the meantime, an alternative approach based on the use of site-specific recombination systems has been developed which enables integration into previously engineered genomic sites (site-specific integration). Follow-up studies have validated the efficacy of the site-specific integration technology in generating transgenic events with a predictable range and stability of expression through successive generations, which are critical features of reliable and practically useful transgenic lines. Any DNA delivery methods can be used for site-specific integration; however, best efficiency is mostly obtained with direct DNA delivery methods such as particle bombardment. Although site-specific integration approach provides unique advantages for producing transgenic plants, it is still not a commonly used method. The present article discusses barriers and solutions for making it readily available to both academic research and applicative use.     

Analysis of the Role of Homology Arms in Gene-Targeting Vectors in Human Cells

Random integration of targeting vectors into the genome is the primary obstacle in human somatic cell gene targeting. Non-homologous end-joining (NHEJ), a major pathway for repairing DNA double-strand breaks, is thought to be responsible for most random integration events; however, absence of DNA ligase IV (LIG4), the critical NHEJ ligase, does not significantly reduce random integration frequency of targeting vector in human cells, indicating robust integration events occurring via a LIG4-independent mechanism. To gain insights into the mechanism and robustness of LIG4-independent random integration, we employed various types of targeting vectors to examine their integration frequencies in LIG4-proficient and deficient human cell lines. We find that the integration frequency of targeting vector correlates well with the length of homology arms and with the amount of repetitive DNA sequences, especially SINEs, present in the arms. This correlation was prominent in LIG4-deficient cells, but was also seen in LIG4-proficient cells, thus providing evidence that LIG4-independent random integration occurs frequently even when NHEJ is functionally normal. Our results collectively suggest that random integration frequency of conventional targeting vectors is substantially influenced by homology arms, which typically harbor repetitive DNA sequences that serve to facilitate LIG4-independent random integration in human cells, regardless of the presence or absence of functional NHEJ.     

The macroevolutionary consequences of phenotypic integration: from development to deep time

Phenotypic integration is a pervasive characteristic of organisms. Numerous analyses have demonstrated that patterns of phenotypic integration are conserved across large clades, but that significant variation also exists. For example, heterochronic shifts related to different mammalian reproductive strategies are reflected in postcranial skeletal integration and in coordination of bone ossification. Phenotypic integration and modularity have been hypothesized to shape morphological evolution, and we extended simulations to confirm that trait integration can influence both the trajectory and magnitude of response to selection. We further demonstrate that phenotypic integration can produce both more and less disparate organisms than would be expected under random walk models by repartitioning variance in preferred directions. This effect can also be expected to favour homoplasy and convergent evolution. New empirical analyses of the carnivoran cranium show that rates of evolution, in contrast, are not strongly influenced by phenotypic integration and show little relationship to morphological disparity, suggesting that phenotypic integration may shape the direction of evolutionary change, but not necessarily the speed of it. Nonetheless, phenotypic integration is problematic for morphological clocks and should be incorporated more widely into models that seek to accurately reconstruct both trait and organismal evolution.