Transfer of foreign genes into intact maize cells with high-velocity microprojectiles

This report describes a process for delivering foreign genes into maize cells that does not require the removal of cell walls and is capable of delivering DNA into embryogenic and nonembryogenic tissues. Plasmid harboring a chimeric chloramphenicol acetyltransferase (CAT) gene was adsorbed to the surface of microscopic tungsten particles (microprojectiles). These microprojectiles were then accelerated to velocities sufficient for penetrating the cell walls and membranes of maize cells in suspension culture. High levels of CAT activity were consistently observed after bombardment of cell cultures of the cultivar Black Mexican Sweet, which were comparable to CAT levels observed after electroporation of protoplasts. Measurable increases in CAT levels were also observed in two embryogenic cell lines after bombardment. Gene expression was observed only when an intron from the alcohol dehydrogenase 1 gene of maize was ligated between the 35S promoter and the CAT coding region. CAT activity was detected in cell cultures bombarded with microprojectiles with an average diameter of 1.2 μm, but not after bombardment with microprojectiles 0.6 or 2.4 μm in diameter. Bombarding the same sample several times was found to markedly enhance CAT activity. These results demonstrate that the particle bombardment process can be used to deliver foreign DNA into intact cells of maize. Because this process circumvents the difficulties associated with regenerating whole plants from protoplasts, the particle bombardment process may provide significant advantages over existing DNA delivery methods for the production of transgenic maize plants. In addition, the process should be of value for studying transient and stable gene expression within intact cells and tissues.     

Inheritance and expression of foreign genes in transgenic soybean plants

DNA-coated gold particles were introduced into meristems of immature soybean seeds using electric discharge particle acceleration to produce transgenic fertile soybean plants. The lineages of integrated foreign DNA in two independently transformed plants were followed in the first (R₁) and second (R₂) generation of self-pollinated progeny. One plant (4615) was transformed with the Escherichia coli genes for β-glucuronidase and neomycin phosphotransferase II; the other (3993) was transformed only with the gene for β-glucuronidase. Segregation ratios for the introduced gene(s) were approximately 3:1 for plant 4615 and 1:1 for plant 3993 in the R₁ generation. DNA analysis showed 100% concordance between presence of the foreign gene sequences and enzyme activity. Moreover, all copies of the foreign genes are inherited as a unit in each plant. Plant 3993 segregated in a 1:1 ratio in the R₂ generation. R₁ plants derived from plant 4615, which expressed both genes, gave either 100% or 3:1 expression of both genes in the R₂ generation, demonstrating recovery of both homozygous and heterozygous R₁ plants. Our results show that foreign DNA introduced into soybean plants using electric discharge particle acceleration can be inherited in a Mendelian manner. Results also demonstrate cotransformation of tandem markers and show that both markers are inherited as closely linked genes in subsequent generations. These results indicate that whole plants can be derived from single transformed cells by a de novo organogenic pathway.     

Adenovirus-mediated gene transfer and expression of human β-glucuronidase gene in the liver, spleen, and central nervous system in mucopolysaccharidosis type VII mice

Mucopolysaccharidosis type VII (Sly syndrome) is a lysosomal storage disease caused by inherited deficiency of the lysosomal enzyme β-glucuronidase. A murine model of this disorder has been well characterized and used to study a number of forms of experimental therapies, including gene therapy. We produced recombinant adenovirus that expresses human β-glucuronidase and administered this recombinant adenovirus to β-glucuronidase-deficient mice intravenously. The β-glucuronidase activities in liver and spleen were elevated to 40% and 20%, respectively, of the heterozygote enzymatic level at day 16. Expression persisted for at least 35 days. Pathological abnormalities of these tissues were also improved, and the elevated levels of urinary glycosaminoglycans were reduced in treated mice. However, the β-glucuronidase activity in kidney and brain was not significantly increased. After administration of the recombinant adenovirus directly into the lateral ventricles of mutant mice, the β-glucuronidase activity in crude brain homogenates increased to 30% of heterozygote activity. Histochemical demonstration of β-glucuronidase activity in brain revealed that the enzymatic activity was mainly in ependymal cells and choroid. However, in some regions, the adenovirus-mediated gene expression was also evident in brain parenchyma associated with vessels and in the meninges. These results suggest that adenovirus-mediated gene delivery might improve the central nervous system pathology of mucopolysaccharidosis in addition to correcting visceral pathology.     

Regulation of anthocyanin biosynthetic genes introduced into intact maize tissues by microprojectiles

We have employed microprojectiles to deliver genes involved in anthocyanin biosynthesis to cells within intact aleurone and embryo tissues of maize. Clones of the A1 or Bz1 genes were introduced into aleurone tissue that lacked anthocyanins due to mutations of the endogenous A1 or Bz1 gene. Following bombardment, cells within the aleurone developed purple pigmentation, indicating that the mutation in the a1 or bz1 genotypes was corrected by the introduced gene. To analyze the expression of these genes in different genetic backgrounds, chimeric genes containing the 5′ and 3′ regions of the A1 or Bz1 genes fused to a luciferase coding region were constructed. These constructs were introduced into aleurones of genotypes carrying either dominant or recessive alleles of the C1 and R genes, which are known to regulate anthocyanin production. Levels of luciferase activity in permissive backgrounds (C1, R) were 30- to 200-fold greater than those detected in tissue carrying one or both of the recessive alleles (c1, r) of these genes. These results show that genes delivered to intact tissues by microprojectiles are regulated in a manner similar to the endogenous genes. The transfer of genes directly to intact tissues provides a rapid means for analyzing the genetic and tissue-specific regulation of gene expression.     

Mature monocytic cells enter tissues and engraft

The goal of this study was to identify the circulating cell that is the immediate precursor of tissue macrophages. ROSA 26 marrow mononuclear cells (containing the β-geo transgene that encodes β-galactosidase and neomycin resistance activities) were cultured in the presence of macrophage colony-stimulating factor and flt3 Ligand for 6 days to generate monocytic cells at all stages of maturation. Expanded monocyte cells (EMC), the immature (ER-MP12⁺) and more mature (ER-MP20⁺) subpopulations, were transplanted into irradiated B6/129 F2 mice. β-gal staining of tissue sections from animals 15 min after transplantation demonstrated that the donor cells landed randomly. By 3 h, donor cells in lung and liver were more frequent in animals transplanted with ER-MP20⁺ (more mature) EMC than in animals transplanted with unseparated EMC or fresh marrow mononuclear cells, a pattern that persisted at 3 and 7 days. At 3 days, donor cells were found in spleen, liver, lung, and brain (rarely) as clusters as well as individual cells. By 7 and 14 days, the clusters had increased in size, and the cells expressed the macrophage antigen F4/80, suggesting that further replication and differentiation had occurred. PCR for the neogene was used to quantitate the amount of donor DNA in tissues from transplanted animals and confirmed that ER-MP20⁺ EMC preferentially engrafted. These data demonstrate that a mature monocytic cell gives rise to tissue macrophages. Because these cells can be expanded and manipulated in vitro, they may be a suitable target population for gene therapy of lysosomal storage diseases.     

Generation and reproductive phenotypes of mice lacking estrogen receptor β

Estrogens influence the differentiation and maintenance of reproductive tissues and affect lipid metabolism and bone remodeling. Two estrogen receptors (ERs) have been identified to date, ERα and ERβ. We previously generated and studied knockout mice lacking estrogen receptor α and reported severe reproductive and behavioral phenotypes including complete infertility of both male and female mice and absence of breast tissue development. Here we describe the generation of mice lacking estrogen receptor β (ERβ −/−) by insertion of a neomycin resistance gene into exon 3 of the coding gene by using homologous recombination in embryonic stem cells. Mice lacking this receptor develop normally and are indistinguishable grossly and histologically as young adults from their littermates. RNA analysis and immunocytochemistry show that tissues from ERβ −/− mice lack normal ERβ RNA and protein. Breeding experiments with young, sexually mature females show that they are fertile and exhibit normal sexual behavior, but have fewer and smaller litters than wild-type mice. Superovulation experiments indicate that this reduction in fertility is the result of reduced ovarian efficiency. The mutant females have normal breast development and lactate normally. Young, sexually mature male mice show no overt abnormalities and reproduce normally. Older mutant males display signs of prostate and bladder hyperplasia. Our results indicate that ERβ is essential for normal ovulation efficiency but is not essential for female or male sexual differentiation, fertility, or lactation. Future experiments are required to determine the role of ERβ in bone and cardiovascular homeostasis.     

DNA polymerase ζ cooperates with polymerases κ and ι in translesion DNA synthesis across pyrimidine photodimers in cells from XPV patients

Human cells tolerate UV-induced cyclobutane pyrimidine dimers (CPD) by translesion DNA synthesis (TLS), carried out by DNA polymerase η, the POLH gene product. A deficiency in DNA polymerase η due to germ-line mutations in POLH causes the hereditary disease xeroderma pigmentosum variant (XPV), which is characterized by sunlight sensitivity and extreme predisposition to sunlight-induced skin cancer. XPV cells are UV hypermutable due to the activity of mutagenic TLS across CPD, which explains the cancer predisposition of the patients. However, the identity of the backup polymerase that carries out this mutagenic TLS was unclear. Here, we show that DNA polymerase ζ cooperates with DNA polymerases κ and ι to carry out error-prone TLS across a TT CPD. Moreover, DNA polymerases ζ and κ, but not ι, protect XPV cells against UV cytotoxicity, independently of nucleotide excision repair. This presents an extreme example of benefit-risk balance in the activity of TLS polymerases, which provide protection against UV cytotoxicity at the cost of increased mutagenic load.     

Directing gene expression to cerebellar granule cells using γ-aminobutyric acid type A receptor α6 subunit transgenes

Expression of the γ-aminobutyric acid type A receptor α6 subunit gene is restricted to differentiated granule cells of the cerebellum and cochlear nucleus. The mechanisms underlying this limited expression are unknown. Here we have characterized the expression of a series of α6-based transgenes in adult mouse brain. A DNA fragment containing a 1-kb portion upstream of the start site(s), together with exons 1–8, can direct high-level cerebellar granule cell-specific reporter gene expression. Thus powerful granule cell-specific determinants reside within the 5′ half of the α6 subunit gene body. This intron-containing transgene appears to lack the cochlear nucleus regulatory elements. It therefore provides a cassette to deliver gene products solely to adult cerebellar granule cells.     

Mislocalization of phosphotransferase as a cause of mucolipidosis III αβ

The lysosomal storage disorder mucolipidosis III αβ is caused by mutations in the αβ subunits of UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (phosphotransferase). This Golgi-localized enzyme mediates the first step in the synthesis of the mannose 6-phosphate recognition marker on lysosomal acid hydrolases, and loss of function results in impaired lysosomal targeting of these acid hydrolases and decreased lysosomal degradation. Here we show that two patient missense mutations, Lys4Gln and Ser15Tyr, in the N-terminal cytoplasmic tail of the α subunit of phosphotransferase impair retention of the catalytically active enzyme in the Golgi complex. This results in mistargeting of the mutant phosphotransferases to lysosomes, where they are degraded, or to the cell surface and release into the medium. The finding that mislocalization of active phosphotransferase is the basis for mucolipidosis III αβ in a subset of patients shows the importance of single residues in the cytoplasmic tail of a Golgi-resident protein for localization to this compartment.Lysosomes are vital for cells to degrade intracellular and endocytosed material. For this reason, defective lysosome biogenesis has detrimental effects on cellular function and is associated with a variety of diseases. Mucolipidosis (ML) III αβ is a lysosomal storage disorder caused by the impaired targeting of newly synthesized acid hydrolases to lysosomes. This results in severely decreased lysosomal degradative capacity and the accumulation of storage material in enlarged lysosomes in many tissues (1).MLIII αβ results from mutations in the GNPTAB gene that encodes the α and β subunits of the enzyme UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (phosphotransferase). Phosphotransferase catalyzes the first step in the generation of the mannose 6-phosphate (Man-6-P) recognition marker on newly synthesized acid hydrolases that directs them to the lysosome. It transfers GlcNAc-1-P to mannose residues on the hydrolases in the cis-Golgi complex. The GlcNAc is then excised by a second enzyme, termed uncovering enzyme, to generate the Man-6-P monoester, which serves as a high-affinity ligand for the Man-6-P receptors. These receptors transport the acid hydrolases from the trans-Golgi network to the endo-lysosomal system, where they function. Phosphotransferase is a heterohexameric enzyme composed of two α, β, and γ subunits (2). The α and β subunits are synthesized as a catalytically inactive αβ precursor of 1,256 amino acids. It is a type 3 transmembrane protein, with a 19-amino acid N-terminal tail (Fig. 1A) and a 21-amino acid C-terminal tail, both of which are cytoplasmically oriented. The αβ subunits undergo a proteolytic cleavage between amino acid Lys928 and Asp929 in the Golgi complex by the site 1 protease to generate the mature, catalytically active α and β subunits (3, 4). These subunits also have the ability to recognize the lysosomal acid hydrolases as specific substrates (5). The γ subunit, encoded by the GNPTG gene, is a soluble glycoprotein of 305 amino acids that enhances the phosphorylation of certain lysosomal enzyme substrates (5).Open in a separate windowFig. 1.Phosphotransferase mutations K4Q and S15Y result in reduced levels of the mature β subunit but do not affect the catalytic activity of the residual protein. (A) Schematic representation of the α (blue) and β (red) subunits of human phosphotransferase, showing the positions of the K4Q and S15Y mutations in the N-terminal tail. TM, transmembrane domain. (B) Phosphotransferase activity toward α-MM was measured in HEK293 cell lysates overexpressing the WT or mutant phosphotransferase αβ subunits (Upper). The relative activity (Lower) shows the activity corrected for the level of mature β subunit relative to WT. Data are average values of 3 independent experiments, error bars represent SDs. *P < 0.01 (Student t test). (C) HEK293 cell lysates expressing WT, K4Q, or S15Y phosphotransferase αβ subunits with a C-terminal V5-tag were subjected to SDS/PAGE and anti-V5 immunoblotting 48 h after transfection (representative blot of 3 independent experiments). The ratio of mature β subunit to the precursor is reduced in the case of the mutants compared with WT.A number of missense mutations have been detected in individuals with MLIII αβ. In this study, the effect of two missense mutations in the N-terminal cytoplasmic tail of the α subunit of phosphotransferase, Lys4Gln (K4Q) and Ser15Tyr (S15Y) (6–8), was investigated (Fig. 1A). These mutations did not directly affect the catalytic function of the enzyme. Instead, they resulted in the failure of the mutant proteins to be retained in the Golgi complex, leading to enhanced degradation in lysosomes or release into the culture medium. As a result, the activity of these mutants in cells was significantly decreased. Thus, mislocalization of phosphotransferase was shown to be the underlying cause of the MLIII αβ phenotype in patients with K4Q and S15Y mutations. The finding that these two residues are critical for localization of phosphotransferase to the cis-Golgi complex may allow for a better understanding of the mechanism that localizes Golgi-resident enzymes to this compartment.     

T cell receptor gene deletion circles identify recent thymic emigrants in the peripheral T cell pool

Progenitor cells undergo T cell receptor (TCR) gene rearrangements during their intrathymic differentiation to become T cells. Rearrangements of the variable (V), diversity (D), and joining (J) segments of the TCR genes result in deletion of the intervening chromosomal DNA and the formation of circular episomes as a byproduct. Detection of these extrachromosomal excision circles in T cells located in the peripheral lymphoid tissues has been viewed as evidence for the existence of extrathymic T cell generation. Because all of the T cells in chickens apparently are generated in the thymus, we have employed this avian model to determine the fate of the V(D)J deletion circles. In normal animals we identified TCR Vγ-Jγ and Vβ-Dβ deletion circles in the blood, spleen, and intestines, as well as in the thymus. Thymectomy resulted in the gradual loss of these DNA deletion circles in all of the peripheral lymphoid tissues. A quantitative PCR analysis of Vγ1-Jγ1 and Vβ1-Dβ deletion circles in splenic γδ and Vβ1⁺ αβ T cells indicated that their numbers progressively decline after thymectomy with a half-life of approximately 2 weeks. Although TCR deletion circles therefore cannot be regarded as reliable indicators of in situ V(D)J rearrangement, measuring their levels in peripheral T cell samples can provide a valuable index of newly generated T cells entering the T cell pool.     

In vivo selection of genetically modified erythroblastic progenitors leads to long-term correction of beta-thalassemia

Gene therapy for β-thalassemia requires stable transfer of a β-globin gene into hematopoietic stem cells (HSCs) and high and regulated hemoglobin expression in the erythroblastic progeny. We developed an erythroid-specific lentiviral vector driving the expression of the human β-globin gene from a minimal promoter/enhancer element containing two hypersensitive sites from the β-globin locus control region. Transplantation of transduced HSCs into thalassemic mice leads to stable and long-term correction of anemia with all red blood cells expressing the transgene. A frequency of 30–50% of transduced HSCs, harboring an average vector copy number per cell of 1, was sufficient to fully correct the thalassemic phenotype. In the mouse model of Cooley's anemia transplantation of transduced cells rescues lethality, leading to either a normal or a thalassemia intermedia phenotype. We show that genetically corrected erythroblasts undergo in vivo selection with preferential survival of progenitors harboring proviral integrations in genome sites more favorable to high levels of vector-derived expression. These data provide a rationale for a gene therapy approach to β-thalassemia based on partially myeloablative transplantation protocols.     

Characteristics of DNA repair induced by DNA polymerase β in hepatoma cells after γ-ray irradiation

AIM: To investigate the effects of DNA repair induced by DNA polymerase β in hepatoma cells after γ-ray irradiation.METHODS: Cell nuclei were prepared from mouse model (SMMC LTNM), in which human hepatoma cells are transplanted on nude mice. The nuclei were then irradiated with ⁶⁰Co-γ rays at different dose levels or dose rates. A selective inhibitor test was then used to detect the effects of the radiation on DNA repair using N-ethylmaleimide (NEM) and ddTTP as selective inhibitors to DNA polymerases γ and β respectively.RESULTS: ³H-TTP incorporation into irradiated nuclei or calf thymus DNA was significantly higher than that the rate at which it is incorporated into non-irradiated nuclei when either DNA polymerase β or γ was inhibited. When both NEM and ddTTP are present, the ³H-TTP incorporation in irradiated DNA was not significantly different from the non-irradiated nuclei. Furthermore, ³H-TTP incorporation into DNA of SMMC-LTNM hepatoma nuclei was higher than that of normal hepatocyte nuclei (P < 0.01). This suggests that DNA repair induced by DNA polymerase β was more active in hepatoma cell nuclei than in normal hepatocyte nuclei.CONCLUSION: DNA polymerase β may be more responsive to DNA damage in some tumor cells than that in normal cells, which may facilitate the cells to repair DNA damages from radiation more efficiently.     

Isolation and characterization of a gene for a major light-harvesting polypeptide from Cyanophora paradoxa

Antibodies raised against mixtures of phycobilisome polypeptides from the eukaryotic alga Cyanidium caldarium were used in an immunological screen to detect expression of phycobiliprotein genes in an Escherichia coli library containing segments of plastid (chloroplast, cyanelle) DNA from another eukaryotic alga, Cyanophora paradoxa. The four candidate clones obtained were mapped by restriction analysis and found to be overlapping. The clone with the smallest insert (1.4 kilobases) was partially sequenced and a coding region similar to the carboxyl terminus of the phycobiliprotein subunit β-phycocyanin was found. The coding region for the β-phycocyanin gene in C. paradoxa has been mapped to the small single copy region on the cyanelle genome, and its orientation has been determined. A short probe unique to a conserved chromophore binding site shared by at least two phycobiliprotein subunits has now been generated from the carboxyl terminus of the β-phycocyanin gene. This probe may be useful in identifying specific phycobiliprotein subunit genes, β-phycocyanin, β-phycoerythrocyanin, and possibly β-phycoerythrin, in other eukaryotic algae and in prokaryotic cyanobacteria.     

Rapid deletion of rearranged T cell antigen receptor (TCR) Vα-Jα segment by secondary rearrangement in the thymus: Role of continuous rearrangement of TCR α chain gene and positive selection in the T cell repertoire formation

A rearranged T cell receptor (TCR) Vα and Jα gene from a cytochrome c-specific T cell hybridoma was introduced into the genomic Jα region. The introduced TCR α chain gene is expressed in a majority of CD3 positive and CD4 CD8 double-negative immature thymocytes. However, only a few percent of the double-positive and single-positive thymocytes express this TCR α chain. This decrease is caused by a rearrangement of TCR α chain locus, which deletes the introduced TCR gene. Analysis of the mice carrying the introduced TCR α chain and the transgenic TCR β chain from the original cytochrome c-specific T cell hybridoma revealed that positive selection efficiently rescues double-positive thymocytes from the loss of the introduced TCR α chain gene. In the mice with negatively selecting conditions, T cells expressing the introduced TCR αβ chains were deleted at the double-positive stage. However, a large number of thymocytes escape negative selection by using an endogenous TCR α chain created by secondary rearrangement maintaining normal thymocyte development. These results suggest that secondary rearrangements of the TCR α chain gene play an important role in the formation of the T cell repertoire.     

Nuclear localization signal binding protein from Arabidopsis mediates nuclear import of Agrobacterium VirD2 protein

T-DNA nuclear import is a central event in genetic transformation of plant cells by Agrobacterium. Presumably, the T-DNA transport intermediate is a single-stranded DNA molecule associated with two bacterial proteins, VirD2 and VirE2, which most likely mediate the transport process. While VirE2 cooperatively coats the transported single-stranded DNA, VirD2 is covalently attached to its 5′ end. To better understand the mechanism of VirD2 action, a cellular receptor for VirD2 was identified and its encoding gene cloned from Arabidopsis. The identified protein, designated AtKAPα, specifically bound VirD2 in vivo and in vitro. VirD2–AtKAPα interaction was absolutely dependent on the carboxyl-terminal bipartite nuclear localization signal sequence of VirD2. The deduced amino acid sequence of AtKAPα was homologous to yeast and animal nuclear localization signal-binding proteins belonging to the karyopherin α family. Indeed, AtKAPα efficiently rescued a yeast mutant defective for nuclear import. Furthermore, AtKAPα specifically mediated transport of VirD2 into the nuclei of permeabilized yeast cells.