High-efficiency homologous recombination in the oil-producing alga Nannochloropsis sp

Algae have reemerged as potential next-generation feedstocks for biofuels, but strain improvement and progress in algal biology research have been limited by the lack of advanced molecular tools for most eukaryotic microalgae. Here we describe the development of an efficient transformation method for Nannochloropsis sp., a fast-growing, unicellular alga capable of accumulating large amounts of oil. Moreover, we provide additional evidence that Nannochloropsis is haploid, and we demonstrate that insertion of transformation constructs into the nuclear genome can occur by high-efficiency homologous recombination. As examples, we generated knockouts of the genes encoding nitrate reductase and nitrite reductase, resulting in strains that were unable to grow on nitrate and nitrate/nitrite, respectively. The application of homologous recombination in this industrially relevant alga has the potential to rapidly advance algal functional genomics and biotechnology.     

Gene targeting by homologous recombination in mouse zygotes mediated by zinc-finger nucleases

Gene targeting by homologous recombination in embryonic stem cells is extensively used to generate specific mouse mutants. However, most mammalian species lack tools for targeted gene manipulation. Since double-strand breaks strongly increase the rate of homologous recombination at genomic loci, we explored whether gene targeting can be directly performed in zygotes by the use of zinc-finger nucleases. Here we report that gene targeting is achieved in 1.7–4.5% of murine one-cell embryos upon the coinjection of targeting vectors with zinc-finger nucleases, without preselection. These findings enable the manipulation of the mammalian germ line in a single step in zygotes, independent of ES cells.Gene targeting by homologous recombination in ES cells is widely used to mutate and modify the mouse genome. Thereby the mouse is established as the most commonly used animal model system (1). With the exception of mice, most other mammalian species lack tools for targeted gene manipulation because functional ES cell lines are not established. Gene targeting in somatic cells combined with nuclear transfer has been used as an alternative for the engineering of livestock species, but represents an elaborate task (2, 3).Zinc-finger nucleases (ZFN) link a DNA binding domain of the zinc-finger type to the nuclease domain of Fok I and enable the induction of double-strand breaks (DSBs) at preselected genomic sites (4). DSBs closed by the error-prone, nonhomologous end-joining (NHEJ) DNA repair pathway frequently exhibit nucleotide deletions and insertions at the cleavage site. This technology has been applied to introduce knockout mutations into the germ line of rats and zebrafish by the expression of ZFNs in early embryos that target coding sequences (5–8).It has been initially shown by using the yeast homing endonuclease I-SceI that the induction of DSBs at genomic insertions of I-SceI recognition sites increases the rate of homologous recombination (HR) in mammalian cells by several orders of magnitude (9). Artificially designed ZFNs further increased the ability to generate site-specific double-strand breaks in endogenous genes, without the requirement to introduce artificial nuclease recognition sites. Following this principle, ZFNs have been used to achieve efficient homologous recombination of gene targeting vectors with various endogenous loci in cultured and primary mammalian cells (10–13).Zygotes provide a logical substrate for the single-step modification of the mammalian germ line. Whereas pronuclear DNA injection is routinely used to generate transgenic mice, rats, and other mammals by random genomic integration of transgenes (14–16), gene targeting in zygotes is not established due to the low rate of spontaneous HR. Because the mouse is the best established mammalian genetic model system, we explored whether targeted mutants can be directly obtained through ZFN-assisted homologous recombination in zygotes, upon the coinjection of ZFN mRNAs and gene targeting vectors (Fig. 1).Open in a separate windowFig. 1.Principle of ZFN-assisted gene targeting in zygotes. Zygotes collected from wild-type mice are coinjected into the pronucleus and cytoplasm with DNA of a gene targeting vector and mRNAs for the expression of a pair of gene specific zinc-finger nucleases (ZFN1/2). HR of the targeting vector with the target site results in a knockout (KO) or knock-in (KI) allele. Manipulated zygotes are subsequently transferred into pseudopregnant females to recover mutant mice.     

Manipulating the mammalian genome by homologous recombination

Gene targeting in mammalian cells has proven invaluable in biotechnology, in studies of gene structure and function, and in understanding chromosome dynamics. It also offers a potential tool for gene-therapeutic applications. Two limitations constrain the current technology: the low rate of homologous recombination in mammalian cells and the high rate of random (nontargeted) integration of the vector DNA. Here we consider possible ways to overcome these limitations within the framework of our present understanding of recombination mechanisms and machinery. Several studies suggest that transient alteration of the levels of recombination proteins, by overexpression or interference with expression, may be able to increase homologous recombination or decrease random integration, and we present a list of candidate genes. We consider potentially beneficial modifications to the vector DNA and discuss the effects of methods of DNA delivery on targeting efficiency. Finally, we present work showing that gene-specific DNA damage can stimulate local homologous recombination, and we discuss recent results with two general methodologies--chimeric nucleases and triplex-forming oligonucleotides--for stimulating recombination in cells.     

Arabidopsis thaliana mutants altered in homologous recombination

Homologous recombination contributes both to the generation of allelic diversity and to the preservation of genetic information. In plants, a lack of suitable experimental material has prevented studies of the regulatory and enzymatic aspects of recombination in somatic and meiotic cells. We have isolated nine Arabidopsis thaliana mutants hypersensitive to x-ray irradiation (xrs) and examined their recombination properties. For the three xrs loci described here, single recessive mutations were found to confer simultaneous hypersensitivities to the DNA-damaging chemicals mitomycin C (MMC^s) and/or methyl methanesulfonate (MMS^s) and alterations in homologous recombination. Mutant xrs9 (Xray^s, MMS^s) is reduced in both somatic and meiotic recombination and resembles yeast mutants of the rad52 epistatic group. xrs11 (Xray^s, MMC^s) is deficient in the x-ray-mediated stimulation of homologous recombination in somatic cells in a manner suggesting a specific signaling defect. xrs4 (Xray^s, MMS^s, MMC^s) has a significant deficiency in somatic recombination, but this is accompanied by meiotic hyper-recombination. A corresponding phenotype has not been reported in other systems and thus this indicates a novel, plant-specific regulatory circuit linking mitotic and meiotic recombination.     

Gene targeting in the mouse: advances in introduction of transgenes into the genome by homologous recombination

The ability to stably introduce genes into the germline of animals provides a powerful means to address the genetic basis of physiology. Introduction of genes to generate transgenic animals has facilitated the development of complex genetic models of disease, as well as the in vivo study of gene function. However, one drawback of traditional transgenic technologies in which genes are microinjected into early-stage embryos is that there is little control over where and in how many copies genes are introduced into the genome. The development of animal transgenic technologies, which take advantage of homologous recombination mechanisms and the manipulation of embryonic stem (ES) cells, allows investigators to target and alter specific loci. In mouse transgenic systems, a plethora of sophisticated gene-targeting strategies now permit investigators to manipulate the genome in ways that essentially allow one to introduce virtually any desired change into the genome. Fur-thermore, when coupled with systems that allow for conditional gene expression, these gene-targeting strategies allow both temporal and tissue specific control of alterations to the genome. In the present review we briefly discuss some of the more recent gene-targeting strategies that have been developed to address the limitations of traditional animal transgenesis.     

Integration of Agrobacterium tumefaciens T-DNA in the Saccharomyces cerevisiae genome by illegitimate recombination

Agrobacterium tumefaciens can transfer part of its Ti plasmid, the T-DNA, to plant cells where it integrates into the nuclear genome via illegitimate recombination. Integration of the T-DNA results in small deletions of the plant target DNA, and may lead to truncation of the T-DNA borders and the production of filler DNA. We showed previously that T-DNA can also be transferred from A. tumefaciens to Saccharomyces cerevisiae and integrates into the yeast genome via homologous recombination. We show here that when the T-DNA lacks homology with the S. cerevisiae genome, it integrates at random positions via illegitimate recombination. From 11 lines the integrated T-DNA was cloned back to Escherichia coli along with yeast flanking sequences. The T-DNA borders and yeast DNA flanking the T-DNA were sequenced and characterized. It was found that T-DNA integration had resulted in target DNA deletions and sometimes T-DNA truncations or filler DNA formation. Therefore, the molecular mechanism of illegitimate recombination by which T-DNA integrates in higher and lower eukaryotes seems conserved.     

Genetic recombination of poliovirus in a cell-free system

Genetic recombination of plus-strand RNA viruses is an important process for promoting genetic variation. By using genetically marked poliovirus RNAs, we have demonstrated that genetic recombination can occur in a cell-free system that generates infective virus from added poliovirus RNA. Recombinant polioviruses were isolated, and the region of crossing over was roughly mapped. Recombinants could be isolated even under conditions where the yield of viruses from one of the parental RNAs was depressed to levels comparable to or less than the yield of recombinant viruses, an observation suggesting that only one of the recombining RNAs needs to be replication-competent. The generation of poliovirus recombinants in a cell-free system offers new possibilities for studying recombination and evolution of RNA viruses.     

Position- and orientation-independent activity of the Schizosaccharomyces pombe meiotic recombination hot spot M26

The activity of the M26 meiotic recombination hot spot of Schizosaccharomyces pombe depends on the presence of the heptamer 5′-ATGACGT-3′. Transplacement of DNA fragments containing the ade6-M26 gene to other chromosomal loci has previously demonstrated that the heptamer functions in some, but not all, transplacements, suggesting that hot spot activity depends on chromosomal context. In this study, hot spot activity was tested in the absence of gross DNA changes by using site-directed mutagenesis to create the heptamer sequence at novel locations in the genome. When created by mutagenesis of 1–4 bp in the ade6 and ura4 genes, the heptamer was active as a recombination hot spot, in an orientation-independent manner, at all locations tested. Thus, the heptamer sequence can create an active hot spot in other chromosomal contexts, provided that the gross chromosomal structure is not altered; this result is consistent with the hypothesis that a specific higher-order chromatin structure is required for M26 hot spot activity.     

Gene transfer of Fas ligand induces tumor regression in vivo

The Fas–Fas ligand (FasL) system plays an important role in the induction of lymphoid apoptosis and has been implicated in the suppression of immune responses. Herein, we report that gene transfer of FasL inhibits tumor cell growth in vivo. Although such inhibition is expected in Fas⁺ tumor cell lines, marked regression was unexpectedly observed after FasL gene transfer into the CT26 colon carcinoma that does not express Fas. Infection by an adenoviral vector encoding FasL rapidly eliminated tumor masses in the Fas⁺ Renca tumor by inducing cell death, whereas the elimination of Fas⁻ CT26 cells was mediated by inflammatory cells. Analysis of human malignancies revealed Fas, but not FasL, expression in a majority of tumors and susceptibility to FasL in most Fas⁺ cell lines. These findings suggest that gene transfer of FasL generates apoptotic responses and induces potent inflammatory reactions that can be used to induce the regression of malignancies.     

The hotspot conversion paradox and the evolution of meiotic recombination

Studies of meiotic recombination have revealed an evolutionary paradox. Molecular and genetic analysis has shown that crossing over initiates at specific sites called hotspots, by a recombinational-repair mechanism in which the initiating hotspot is replaced by a copy of its homolog. We have used computer simulations of large populations to show that this mechanism causes active hotspot alleles to be rapidly replaced by inactive alleles, which arise by rare mutation and increase by recombination-associated conversion. Additional simulations solidified the paradox by showing that the known benefits of recombination appear inadequate to maintain its mechanism. Neither the benefits of accurate segregation nor those of recombining flanking genes were sufficient to preserve active alleles in the face of conversion. A partial resolution to this paradox was obtained by introducing into the model an additional, nonmeiotic function for the sites that initiate recombination, consistent with the observed association of hotspots with functional sites in chromatin. Provided selection for this function was sufficiently strong, active hotspots were able to persist in spite of frequent conversion to inactive alleles. However, this explanation is unsatisfactory for two reasons. First, it is unlikely to apply to obligately sexual species, because observed crossover frequencies imply maintenance of many hotspots per genome, and the viability selection needed to preserve these would drive the species to extinction. Second, it fails to explain why such a genetically costly mechanism of recombination has been maintained over evolutionary time. Thus the paradox persists and is likely to be resolved only by significant changes to the commonly accepted mechanism of crossing over.     

Mechanisms of spectral tuning in the mouse green cone pigment

Diversification of cone pigment spectral sensitivities during evolution is a prerequisite for the development of color vision. Previous studies have identified two naturally occurring mechanisms that produce variation among vertebrate pigments by red-shifting visual pigment absorbance: addition of hydroxyl groups to the putative chromophore binding pocket and binding of chloride to a putative extracellular loop. In this paper we describe the use of two blue-shifting mechanisms during the evolution of rodent long-wave cone pigments. The mouse green pigment belongs to the long-wave subfamily of cone pigments, but its absorption maximum is 508 nm, similar to that of the rhodopsin subfamily of visual pigments, but blue-shifted 44 nm relative to the human red pigment, its closest homologue. We show that acquisition of a hydroxyl group near the retinylidene Schiff base and loss of the chloride binding site mentioned above fully account for the observed blue shift. These data indicate that the chloride binding site is not a universal attribute of long-wave cone pigments as generally supposed, and that, depending upon location, hydroxyl groups can alter the environment of the chromophore to produce either red or blue shifts.     

Rescue of arrested replication forks by homologous recombination

DNA synthesis is an accurate and very processive phenomenon; nevertheless, replication fork progression on chromosomes can be impeded by DNA lesions, DNA secondary structures, or DNA-bound proteins. Elements interfering with the progression of replication forks have been reported to induce rearrangements and/or render homologous recombination essential for viability, in all organisms from bacteria to human. Arrested replication forks may be the target of nucleases, thereby providing a substrate for double-strand break repair enzyme. For example in bacteria, direct fork breakage was proposed to occur at replication forks blocked by a bona fide replication terminator sequence, a specific site that arrests bacterial chromosome replication. Alternatively, an arrested replication fork may be transformed into a recombination substrate by reversal of the forked structures. In reversed forks, the last duplicated portions of the template strands reanneal, allowing the newly synthesized strands to pair. In bacteria, this reaction was proposed to occur in replication mutants, in which fork arrest is caused by a defect in a replication protein, and in UV irradiated cells. Recent studies suggest that it may also occur in eukaryote organisms. We will review here observations that link replication hindrance with DNA rearrangements and the possible underlying molecular processes.     

Direct isolation of human BRCA2 gene by transformation-associated recombination in yeast

Mutant forms of the BRCA2 gene contribute significantly to hereditary breast cancer. Isolation of the normal and mutant forms of the BRCA2 gene with its natural promoter would greatly facilitate analysis of the gene and its contribution to breast cancer. We have accomplished the direct isolation of the 90-kb gene from total human DNA by transformation-associated recombination in yeast using a small amount of 5′ and 3′ BRCA2 sequence information. Because the entire isolation procedure of a single chromosomal gene could be accomplished in approximately 2 weeks, the transformation-associated recombination cloning approach is readily applicable to studies of chromosome alterations and human genetic diseases.     

Double strand breaks at the HIS2 recombination hot spot in Saccharomyces cerevisiae

Double strand breaks (DSBs) have been found at several meiotic recombination hot spots in Saccharomyces cerevisiae; more global studies have found that they occur at many places along several yeast chromosomes during meiosis. Indeed, the number of breaks found is consistent with the number of recombination events predicted from the genetic map. We have previously demonstrated that the HIS2 gene is a recombination hot spot, exhibiting a high frequency of gene conversion and associated crossing over. This paper shows that DSBs occur in meiosis at a site in the coding region and at a site downstream of the HIS2 gene and that the DSBs are dependent upon genes required for recombination. The frequency of DSBs at HIS2 increases when the gene conversion frequency is increased by alterations in the DNA around HIS2, and vice versa. A deletion that increases both DSBs and conversion can stimulate both when heterozygous; that is, it is semidominant and acts to stimulate DSBs in trans. These data are consistent with the view that homologous chromosomes associate with each other before the formation of the DSBs.     

Repeat expansion by homologous recombination in the mouse germ line at palindromic sequences

Genetic instability can be induced by unusual DNA structures and sequence repeats. We have previously demonstrated that a large palindrome in the mouse germ line derived from transgene integration is extremely unstable and undergoes stabilizing rearrangements at high frequency, often through deletions that produce asymmetry. We have now characterized other palindrome rearrangements that arise from complex homologous recombination events. The structure of the recombinants is consistent with homologous recombination occurring by a noncrossover gene conversion mechanism in which a break induced in the palindrome promotes homologous strand invasion and repair synthesis, similar to mitotic break repair events reported in mammalian cells. Some of the homologous recombination events led to expansion in the size of the palindromic locus, which in the extreme case more than doubled the number of repeats. These results may have implications for instability observed at naturally occurring palindromic or quasipalindromic sequences.     

Stimulation of homologous recombination in plants by expression of the bacterial resolvase RuvC

Targeted gene disruption exploits homologous recombination (HR) as a powerful reverse genetic tool, for example, in bacteria, yeast, and transgenic knockout mice, but it has not been applied to plants, owing to the low frequency of HR and the lack of recombinogenic mutants. To increase the frequency of HR in plants, we constructed transgenic tobacco lines carrying the Escherichia coli RuvC gene fused to a plant viral nuclear localization signal. We show that RuvC, encoding an endonuclease that binds to and resolves recombination intermediates (Holliday junctions) is properly transcribed in these lines and stimulates HR. We observed a 12-fold stimulation of somatic crossover between genomic sequences, a 11-fold stimulation of intrachromosomal recombination, and a 56-fold increase for the frequency of extrachromosomal recombination between plasmids cotransformed into young leaves via particle bombardment. This stimulating effect may be transferred to any plant species to obtain recombinogenic plants and thus constitutes an important step toward gene targeting.     

Generation of hybrid human immunodeficiency virus by homologous recombination.

Human immunodeficiency virus (HIV) type 1, isolated from diverse sources, exhibits genomic diversity. The mechanisms by which the genomic diversity takes place in individuals exposed to multiple virus isolates is yet to be elucidated. Genetic variation, in general, might result from mutagenic events such as point mutations, rearrangements (insertions and deletions), and recombination. In an attempt to evaluate the process of genetic diversity, we designed experiments to analyze recombination between HIV DNAs by using DNA transfection in cell cultures. Here we report the successful recombination between truncated HIV proviral DNAs with an overlap homology of 53 base pairs that leads to the formation of viable hybrid virus. Recombination was also seen between exogenous DNA introduced into cells and homologous HIV sequences resident in the cells. These results indicate that recombination among various HIV isolates may play a significant role in the generation of genetic diversity of HIV. Further, the method used here enables the construction of hybrid HIV genomes to identify the viral determinants responsible for tropism, replication, and cytopathic effects.