Humoral autoimmunity to basement membrane antigens is regulated in C57BL/6 and MRL/MpJ mice transgenic for anti-laminin Ig receptors

Basement membrane proteins are targeted in organ-limited and systemic autoimmune nephritis, yet little is known about the origin or regulation of immunity to these complex extracellular matrices. We used mice transgenic for a nephrotropic systemic lupus erythematosus (SLE) Ig H chain to test the hypothesis that humoral immunity to basement membrane is actively regulated. The LamH-Cmu Ig H chain transgene combines with diverse L chains to produce nephrotropic Ig reactive with murine laminin alpha1. To determine the fate of transgene-bearing B cells in vivo, transgenic mice were outcrossed onto nonautoimmune B6 and SLE-prone MRL backgrounds and exposed to potent mitogen or Ag in adjuvant. In this work we demonstrate that transgenic autoantibodies are absent in serum from M6 and M29 lineage transgenic mice and transgenic B cells hypoproliferate and fail to increase Ig production upon exposure to endotoxin or when subjected to B cell receptor cross-linking. Administration of LPS or immunization with autologous or heterologous laminin, maneuvers that induce nonoverlapping endogenous anti-laminin IgG responses, fails to induce a transgenic anti-laminin response. The marked reduction in splenic B cell number suggests that selected LamH-Cmu H chain and endogenous L chain combinations generate autospecificities that lead to B cell deletion. It thus appears that SLE-like anti-laminin B cells have access to and engage a tolerizing self-Ag in vivo. Failure to induce autoimmunity by global perturbations in immune regulation introduced by the MRL autoimmune background and exposure to potent environmental challenge suggests that humoral immunity to nephritogenic basement membrane epitopes targeted in systemic autoimmunity is tightly regulated.     

The rearranged V(H) domain of a physiologically selected anti-single-stranded DNA antibody as a precursor for formation of IgM and IgG antibodies to diverse antigens

It has been proposed that autoreactivity of modest affinity contributes to positive selection of a preimmunization B cell repertoire, whereas high-affinity autoreactivity leads to negative selection. This hypothesis predicts that a B cell producing a physiologically selected unmutated ssDNA-binding Ab should be a precursor of cells that respond to diverse exogenous Ags. To test this prediction, we prepared transgenic mice bearing the rearranged V(H) domain of an IgM Ab from a nonautoimmune mouse immunized with a DNA-protein complex, poly(dC)-methylated BSA. The Ab, dC1, binds both poly(dC) and ssDNA. It is encoded by V(H) and V(L) gene segments with no mutations, suggesting that the producing cell may have been selected before and activated during immunization. The dC1V(H) transgene was targeted to the IgH locus. In heterozygous mice, on a nonautoimmune C57BL/6 background, the transgene allotype was expressed on B cell surfaces and in serum Ig, but about one-third of B cells expressed the endogenous allele instead. Total serum Ig concentrations were normal and included both transgene- and endogenous gene-coded IgM and IgG. The transgene V(H) D(H)J(H) was expressed in splenic IgM cDNA with few or no mutations, and in IgG cDNA with multiple mutations. The transgene allotype was also expressed in Abs formed on immunization with thyroglobulin, pneumococcal polysaccharide, and ssDNA-methylated BSA. Consistent with the hypothesis, cells with a rearranged autoreactive V(H) domain selected for reactivity with a form of ssDNA did serve as precursors for cells producing IgM and IgG Abs to diverse Ags.     

The organization of immunoglobulin variable kappa chain genes on mouse chromosome 6

One mouse with a known recombination (NAK) at the Igk locus on chromosome 6 and two new recombinants [B6.PL (7 NS) and B6.PL (85NS)] were examined using a series of probes, each of which is specific for a set of immunoglobulin (Ig) Vk genes. Under high stringency conditions, each probe detects from 1 to 19 Bam HI restriction endonuclease fragments (REFs) in genomic DNA by Southern transfer hybridization techniques. Analysis of the REF patterns indicate that the NAK recombination event occurred within the variable region of Igk. The REF patterns of the two B6.PL congenic mice provided two additional recombination events which could be examined. Although some of the REFs had shared mobility among the parental strains, at least 1 and up to 13 polymorphic REFs were present for a given probe among the NZB and AKR parental strains. The results from the NAK mouse indicate that at least some members of Vk4, Vk8, Vk10, and Vk21 were on one side of the recombination event linked to the Lyt-2 ^a and Igk-Efl ^a alleles of AKR, while the Vk9, Vk11, and Vk24 REF patterns came from the NZB parental strain linked to the Igk-Ef2 ^b (Vk1) allele. The two B6.PL congenics produced a refined map on the Lyt-2, Lyt-3 side of the Vk region. The B6.PL (85NS) mice retained the Vk21 REF pattern of the Lyt-2 ^a, Lyt-3 ^a donor strain PL/J, while displaying the C57BL/6 REF pattern for the other Vk gene groups tested. The B6.PL (75NS) mice retained the REF patterns of PL/J for Vk21 and Ef-1, indicating a third recombination. This indicates the Vk gene order is (Lyt-2; Vk21); Ef-1; (Vk4; Vk8; Vk10); and (Vk9; Vk11; Vk24; Ef-2).     

Genetic correlates of autoreactivity and autoreactive potential in human Ig heavy chains

Background

Immature bone marrow B cells are known to have longer CDR3 than mature peripheral B cells, and this genetic characteristic has been shown to correlate with autoreactivity in these early cells. B-cell Central tolerance eliminates these cells, but it is known that autoreactive B cells nevertheless appear commonly in healthy human blood. We examined over 7,300 Ig genes from Genbank, including those annotated by their discoverers as associated with autoreactivity, to determine the genetic correlates of autoreactivity in mature B cells.

Results

We find differential biases in gene segment usage and higher mutation frequency in autoreactivity-associated Ig genes, but the CDR3 lengths do not differ between autoreactive and non-autoreactive Ig genes. The most striking genetic signature of autoreactivity is an increase in the proportion of N-nucleotides relative to germline-encoded nucleotides in CDR3 from autoreactive genes.

Conclusion

We hypothesize that peripheral autoreactivity results primarily from somatic mutation, and that the genetic correlates of autoreactivity in mature B-cells are not the same as those for autoreactivity in immature B cells. What is seen in mature autoreactive B cells are the correlates of autoreactive potential, not of autoreactivity per se. The autoreactive potential is higher for V(D)J rearrangements encoded to a large extent by N-nucleotides rather than by the gene segments that, we posit, have been selected in germline evolution for their suppression of autoreactive potential.     

Genetic chasing of T helper cell idiotype and allotype genes

The present experiments were performed to study whether the genes responsible for the expression of T-cell idiotypes and allotypes could be mapped in relation to immunoglobulin (Ig) heavy chain V- and C-genes. Use was made of our antiserum 5936, which detects idiotypes in B6 anti-B10.BR sera and on Lyt-1⁺, 2.3^–B6 anti-B10.BR T-cell populations, and antiserum 6036, which detects allotypes on Lyt-1⁺, 2.3^–B6 T cells, but which does not react against Ig. The reactivity of these antisera with T cells from (B6 x C3H.OH) x C3H.OH backcross mice and CBA-allotype congenic B6 mice was investigated because 5936 idiotypes and 6036 allotypes appeared to be associated with Igh-1 ^b genes (B6) and not with Igh-1 ^b genes (C3H.OH, CBA). Our results will show, first, that 5936 idiotypes on Lyt-1⁺, 2.3^–B6 anti-B10.BR T cells are synthesized by genes linked to Igh-1 ^b allotype genes and they are situated either within Ig heavy chain V-genes or centromeric to them. Second, our results will show that 6036 allotypes on Lyt-1⁺, 2.3^–B6 T cells are produced by genes also linked to Igh-1 ^b -allotype genes, and the 6036 allotype genes are situated between Ig-VH and prealbumin genes.Abbreviations used in this paper BCGF B cell growth factor - B6 C57B1/6 - C_H constant region of immunoglobulin heavy chain - Con A concanavalin A - FCS fetal calf serum - Id idiotype - Ig immunoglobulin - LPS lipopolysaccharide - M mouse - MHC major histocompatibility complex - MLC mixed lymphocyte culture - MRBC mouse red blood cells - NMS normal mouse serum - NP nitrophenacetyl - NRS normal rabbit serum - PFC plaque forming cell - R rabbit - Tcf T cell factor - Tcr T cell receptor - TNP Trinitrophenol - V_H variable region of Ig heavy chain Definitions of terms used in this paper: T-cell idiotypes, structures on T-cell membranes or released T-cell molecules detected by an anti-idiotypic antiserum (5936) produced against specific immunoglobulin idiotypes. The 5936 T-cell idiotypes are related to the specific binding of IA^k gene products by certain Igh-1^b T cells. T cell allotypes, structures on T-cell membranes or released T-cell molecules detected by an antiserum (6036) produced against 5936 idiotype-bearing T-cell molecules. The 6036 T-cell allotypes are related to the binding by Igh-1^b T cells of all Ia gene products tested, and they are non-cross-reactive with immunoglobulin allotypes.     

Basophils support the survival of plasma cells in mice

We have previously shown that basophils support humoral memory immune responses by increasing B cell proliferation and Ig production as well as inducing a Th2 and B helper phenotype in T cells. Based on the high frequency of basophils in spleen and bone marrow, in this study we investigated whether basophils also support plasma cell survival and Ig production. In the absence of basophils, plasma cells of naive or immunized mice rapidly undergo apoptosis in vitro and produce only low amounts of Igs. In contrast, in the presence of basophils and even more in the presence of activated basophils, the survival of plasma cells is markedly increased and continuous production of Igs enabled. This effect is partially dependent on IL-4 and IL-6 released from basophils. Similar results were obtained when total bone marrow cells or bone marrow cells depleted of basophils were cultured in the presence or absence of substances activating basophils. When basophils were depleted in vivo 6 mo after immunization with an Ag, specific Ig production in subsequent bone marrow cultures was significantly reduced. In addition, depletion of basophils for 18 d in naive mice significantly reduced the number of plasma cells in the spleen. These data indicate that basophils are important for survival of plasma cells in vitro and in vivo.     

Effect of morphine on <Emphasis Type="Italic">Mycobacterium smegmatis</Emphasis> infection in mice and macrophages

The immunomodulatory effects of opioids are known in various infections. However, little is known about the effects of opioids in tuberculosis (TB). In the present study, we report the effects of morphine in Mycobacterium smegmatis infection in mice and macrophages. Morphine exerted a dose-dependent suppression of infection in vivo: 50 and 100 mg/kg morphine exerted significant (P<0.05) suppression whereas 5 mg/kg morphine showed no effect. Analogous to the in vivo effects, incubation of M. smegmatis-infected mouse peritoneal macrophages with morphine (100 μM) showed significant reduction in intramacrophage CFU counts. However, morphine did not show any direct antimycobacterial activity in broth dilution assay upto 100 μM concentration. Further, morphine-induced intramacrophage killing of M. smegmatis was abrogated by naloxone and aminoguanidine indicating the involvement of opioid-receptor activation and nitric oxide production in protective effects of morphine. In conclusion, morphine suppressed the progression of experimental TB in both mice and macrophage models.     

Accumulation of unusual sterile transcripts of TCRβ in mouse hybridoma, murine tumour and non-human primate marmoset tumour

Accumulation of immunoglobulin Ig RNA (from several loci viz., C_H, C_α, J_k-C_k and S_μ during Igμ isotype switching) in B cells and T cell receptor (TCR) RNAs (α, β andγ) in T cells of unusual sizes emanating from germline and rearranged genes were reported to accumulate in human and mouse (and murine too). The precise mechanism and function of these sterile RNA species are yet to be delineated. Similar accumulation of RNA species of unusual sizes were identified with DNA-RNA hybridization and isolation of cDNA employing with DNA and antibody probes in mouse hybridoma, murine tumour, non-human primate marmoset tumour and human leukemic cells.     

Boron-aluminum interactions affect organic acid metabolism more in leaves than in roots of <Emphasis Type="Italic">Citrus grandis</Emphasis> seedlings

Sour pummelo (Citrus grandis) seedlings were irrigated with nutrient solution containing four boron concentrations (i.e., 2.5, 10, 25 and 50 μM H₃BO₃) and two aluminum concentrations [i.e., 0 (-Al) and 1.2 mM AlCl₃ · 6 H₂O (+Al)]. It was found that B did not affect, but Al increased, the Al content in the roots. The Al and citrate contents in the -Al leaves either did not change or slightly increased with increasing B concentration. On the other hand, the Al and citrate contents in the +Al leaves rapidly decreased as B concentration increased from 2.5 to 50 μM, then decreased at the highest B concentration. The Al and citrate contents were higher in the +Al than in the -Al leaves, except for at 25 μM B when they were similar. The leaf malate content did not change in response to B or Al, except for an increase in the +Al leaves and a decrease in the -Al leaves at 2.5 μM B. Similarly, the root malate and citrate contents did not change in response to B with or without Al, except for a decrease in the malate and citrate contents in the +Al roots at 50 μM B and an increase in the citrate content in the -Al roots at 50 μM B. The activities of acid-metabolizing enzymes were less affected by B-Al interactions in the roots than in the leaves.     

Embryo rescue of interspecific hybrids of Brachiaria spp

Age of explant and six different media were evaluated with the objective of regenerating higher numbers of interspecific hybrids between sexual and apomictic Brachiaria. Immature embryos of 7–8, 9–10 and 11–12 days after pollination (DAP), from artificial hybridization between Brachiaria ruziziensis (R) as female parent, and B. brizantha (B) or B. decumbens (D) as male parent, were cultured in modified MS media (M4) – supplemented with different combinations of growth regulators and vitamins. Embryos cultured 9–12 DAP showed high percentage (85–100%) of germination for all the crosses examined. Germination and survival rates varied according to accessions within crosses. Six different media (all modified MS with different growth regulators and vitamins) were tested with the objective of inducing multiple shoots from 7 to 10 DAP embryos, from crosses between R × B. The media M1, supplemented with Kinetin (13.94 μM) and NAA (5.37 μM), and media M3, supplemented with BA (4.44 μM and IAA 2.85 μM), regenerated adventitious shoots and calli about 30–40 days after inoculation. The highest multiplication rate observed was 2.85 shoots per explant in media M1, 60–70 days after culturing. Two other media, M6, supplemented with 2,4-D (13.57 μM) and M2, with 2,4-D (9.05 μM) and BA (8.87 μM) exclusively induced the formation of calli. The described protocols proved to be efficient in regenerating healthy seedlings from immature embryos of interspecific hybrids in Brachiaria. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of Strontium Ranelate on Hydrogen Peroxide-Induced Apoptosis of CRL-11372 Cells

In vitro and in vivo studies have proven strontium to be an osteoinductive trace element. The effect of strontium ranelate (SR) on H₂O₂-induced apoptosis of CRL-11372 cells and optimization of its anti-apoptotic dose were the aims of this study. After 1 h of pretreatment with SR 1 μM, 50 μM, 100 μM, 500 μM, and 1,000 μM concentrations, CRL-11372 osteoblasts were exposed to 100 μM H₂O₂ for periods of 6–12 h. The same experiments were repeated without H₂O₂. The apoptotic index and viability of cells were assessed quantitatively with a fluorescent dye and qualitatively with agarose gel electrophoresis. Concentrations of 1–100 μM of SR with a 6-h treatment and only 1 μM concentration with a 12-h treatment inhibited the apoptotic effect of H₂O₂ on cultured osteoblasts significantly (P < 0.05). SR was shown to inhibit H₂O₂-induced apoptosis of CRL-11372 cells in a dose-dependent manner.     

Expansion of a direct shoot organogenesis system in peanut (Arachis hypogaea L.) to include US cultivars

Agrobacterium-mediated transformation, employing direct shoot organogenesis, allows for mature transgenic plants to be obtained quickly (3–4 mo). In this study, peanut (Arachis hypogaea L.) cultivars Florida-07, Georgia Green, Georgia Brown, New Mexico Valencia A, and VC-2 were selected to test their shoot induction response for use in future transformation experiments. Two types of cotyledon explants were examined, those that previously had an attached embryo axis upon cotyledon separation (explant A) and those that were embryo axis-free upon separation (explant B). Explants were placed onto a shoot induction medium with N ⁶-benzyladenine concentrations ranging from 10–80 μM for Florida-07, Georgia Green, and VC-2; 10–20 μM for Georgia Brown; and 10–640 μM for New Mexico Valencia A. Following a 4-wk culture period, explants were visually rated based on a scale of 1–4, where 1 indicated slight greening, but no growth, and 4 indicated greening, adventitious bud formation, as well as small leaf expansion. A difference in shoot induction was observed for the cotyledon explants examined (P > t = <0.0001). Explant A had greater shoot induction with a visual rating of 1.8 ± 0.1; explant B had a rating of 1.6 ± 0.1 (P > t = <0.0001). Additionally, cultivars responded to the culture conditions differently (cultivar × N ⁶-benzyladenine interaction). Georgia Green on 10 μM N ⁶-benzyladenine produced the most shoot buds (24.6%) and the highest visual rating (2.1), followed by VC-2 on 10 μM N ⁶-benzyladenine (22.1%, 1.8), New Mexico Valencia A on 640 μM N ⁶-benzyladenine (21.4%, 1.8), Georgia Brown on 80 μM N ⁶-benzyladenine (9.0%, 1.7), and Florida-07 on 40 μM N ⁶-benzyladenine (7.1%, 1.8). Of the tested varieties, Georgia Green, New Mexico Valencia A, and VC-2 were best suited for future transformation experiments based on their shoot bud production.     

Analysis of Substrate Specificity of Pig CYP2B22 and CYP2C49 towards Herbicides by Transgenic Rice Plants

We introduced two novel types of pig (Sus scrofa) cytochrome P450, CYP2B22 and CYP2C49, into rice plants (Oryza sativa L. cv. ‘Nipponbare’) to produce herbicide-tolerant plants and to confirm the metabolic activities of the cytochrome P450 species. In germination tests, both types of transgenic plants showed tolerance to various herbicides with different modes of action. CYP2B22 rice plants showed tolerance towards 12 herbicides including chlortoluron (100 μM), amiprofos-methyl (2.5 μM), pendimethalin (10 μM), metolachlor (2.5 μM), and esprocarb (20 μM). CYP2C49 rice plants showed tolerance towards 13 herbicides, including chlortoluron (100 μM), norflurazon (0.5 μM), amiprofos-methyl (2.5 μM), alachlor (0.8 μM), and isoxaben (1 μM). The herbicide tolerance was considered to reflect the substrate specificity of the introduced P450 species. We used ¹⁴C-labeled metolachlor and norflurazon to confirm the P450 activity in the transgenic rice plants. The herbicides were metabolized more quickly in the transgenic rice plants than in the nontransgenic rice plants. Therefore, CYP2B22 and CYP2C49 rice plants became more tolerant to various herbicides than nontransgenic control plants because of accelerated metabolism of the herbicides by the introduced P450 species. Assuming that public and commercial acceptance is forthcoming, these transgenic rice plants may become useful tools for the breeding of herbicide-tolerant crops.     

Development of myelin oligodendrocyte glycoprotein autoreactive transgenic B lymphocytes: receptor editing in vivo after encounter of a self-antigen distinct from myelin oligodendrocyte glycoprotein

We explored mechanisms involved in B cell self-tolerance against brain autoantigens in a double-transgenic mouse model carrying the Ig H-chain (introduced by gene replacement) and/or the L-chain kappa (conventional transgenic) of the mAb 8.18C5, specific for the myelin oligodendrocyte glycoprotein (MOG). Previously, we demonstrated that B cells expressing solely the MOG-specific Ig H-chain differentiate without tolerogenic censure. We show now that double-transgenic (THkappa(mog)) B cells expressing transgenic Ig H- and L-chains are subjected to receptor editing. We show that in adult mice carrying both MOG-specific Ig H- and L-chains, the frequency of MOG-binding B cells is not higher than in mice expressing solely the transgenic Ig H-chain. In fact, in THkappa(mog) double-transgenic mice, the transgenic kappa(mog) L-chain was commonly replaced by endogenous L-chains, i.e., by receptor editing. In rearrangement-deficient RAG-2(-) mice, differentiation of THkappa(mog) B cells is blocked at an immature stage (defined by the B220(low)IgM(low)IgD(-) phenotype), reflecting interaction of the autoreactive B cells with a local self-determinant. The tolerogenic structure in the bone marrow is not classical MOG, because back-crossing THkappa(mog) mice into a MOG-deficient genetic background does not lead to an increase in the proportion of MOG-binding B cells. We propose that an as yet undefined self-Ag distinct from MOG cross-reacts with the THkappa(mog) B cell receptor and induces editing of the transgenic kappa(mog) L-chain in early immature B cells without affecting the pathogenic potential of the remaining MOG-specific B cells. This phenomenon represents a particular form of chain-specific split tolerance.     

Somatic embryogenesis and Agrobacterium-mediated transformation system for scented geraniums (Pelargonium sp. `Frensham')

Plant regeneration via somatic embryogenesis was achieved from leaf petioles of Pelargonium sp. `Frensham' cultured on Murashige and Skoog medium containing 15 μM N⁶-benzyladenine, and 5 μM α-naphthaleneacetic acid (NAA). More than 80% of these somatic embryos converted into plants when isolated and cultured on Murashige and Skoog medium supplemented with 15 μM NAA. Stable transgenic plants were obtained by co-cultivation of the petioles (prior to culture) with Agrobacterium tumefaciens strains LBA4404 (harbouring a binary vector pBI121 carrying the nptII and gus genes) and LBG66 (harbouring a binary plasmid pJQ418 carrying the gus/int:nptII fusion gene). Transformants were selected using kanamycin and transformation was verified by β-glucuronidase histochemical assay and polymerase chain reaction. Southern analysis further confirmed the integration of these genes into the genome of transgenic plants. We report here for the first time, an Agrobacterium-mediated model transformation system coupled with regeneration via somatic embryogenesis for production of transgenics in Pelargonium sp. Received: 20 September 1996 / Accepted: 13 November 1996     

Effects of growth regulators and incubation period on in vitro regeneration of adventitious shoots from gerbera petioles

An effective system for in vitro regeneration of adventitious shoots from callus for the transformation or mutation of gerbera was developed. Callus was produced from petioles of the youngest 3–4 leaves detached from auxillary shoots produced in vitro. Induction medium, on which leaves were incubated over 3 or 6 days, contained 2.3 μM thidiazuron and 0.53 μM α-naphthaleneacetic acid. Explants were than transferred to one of three regeneration media with lower levels of growth regulators. Regeneration was quantified over four (4-weeks each) passages at the time of explant transfer to fresh medium. Direct shoot regeneration occurred during the first 4 weeks, and after these shoots were discarded a semi-compact organogenic callus was produced. Effectiveness of shoot regeneration depended on four criteria: the cultivar (three cultivars were tested), the sequence of passage on regeneration medium, the growth regulators in regeneration medium and the duration of the induction period. Regeneration potential from calli of all cultivars increased from the first to the fourth passage. Duration of the incubation period on induction medium (3 or 6 days) influenced regeneration to varying degrees, depending on the cultivar used and the regeneration medium contents. There were no differences between two of the regeneration media – B, containing 2.2 μM 6-benzyladenine and 0.3 μM indole-3-acetic acid and C, containing 4.4 μM 6-benzyladenine, 4.6 μM zeatin and 0.6 μM indole-3-acetic acid. Cultivar Mariola was the most productive and regenerated more than seven shoots per callus in the fourth passage. Regeneration on medium B was further evaluated on four additional gerbera cultivars. This revised version was published online in June 2006 with corrections to the Cover Date.     

rol-Transgenic Populus tremula: root development, root-borne bud regeneration and in vitro propagation efficiency

 The potential use of the rol genes from Agrobacterium rhizogenes to improve the root system horticultural characteristics was evaluated in transgenic aspen (Populus tremula) plants, harboring the rol genes under their native promoters. Southern blot and RT-PCR analyses confirmed the presence and expression of A. rhizogenes rolC and rolB genes in four different phenotypically selected transgenic clones. Several of the observed phenotypic modifications were related to rol-gene expression and included, in particular, modified root systems. All in vitro-cultured rol-transgenic plants exhibited extensive root formation in a hormone-free medium, as well as a larger root surface area and mass, as compared to a uidA (β-glucuronidase-encoding)-transgenic aspen line and control (non-transformed) plants. Adventitious root formation in stem segments of rol-transgenic plants exhibited very rapid kinetics, resulting in a much shorter rooting time for rol-transgenic stem segments (e.g. 10 days for 80% rooting in rol-transgenic lines T-26 and T-27, as compared to more than 18 days for control non-transformed or uidA-transgenic aspen plants). rol-Transgenic plants maintained the capacity for 100% rooting throughout the year, versus 70–80% rooting in non-transformed plants during the winter. The four rol-transgenic lines exhibited differences in root development; in two of them enhanced root development was accompanied by increased shoot fresh weight. The root:shoot fresh weight ratio was always higher in rol-transgenic lines than in non-transformed plants. In the T-27 rol-transgenic line, the propagation coefficient of shoot-bud regeneration in liquid root culture was almost three times higher than in non-transformed plants. To the best of our knowledge this is the first report on quantitative phenotypic alterations in rol-transgenic woody plants. Received: 1 December 1997 / Accepted: 8 March 1998     

A suicide vector for allelic recombination involving the gene for glutamate 1-semialdehyde aminotransferase in the cyanobacterium Synechococcus PCC 7942

Gabaculine (2,3-dihydro 3-amino benzoic acid) is a potent inhibitor of tetrapyrrole biosynthesis in organisms that use the C₅ pathway for the synthesis of δ-aminolaevulinic acid. Glutamate semialdehyde aminotransferase (GSA-AT), the enzyme catalysing the formation of this key precursor of tetrapyrroles, is normally inhibited by concentrations of gabaculine in the order of 5 μM. However, in Synechococcus 6301 strain GR6, a cyanobacterium that is resistant to 100 μM gabaculine, this enzyme has undergone two changes in structure: a deletion of three amino acids from positions 5 to 7 and the substitution of isoleucine for methionine at position 248. To establish the effect in vivo of these specific changes in the gene for GSA-AT (hemL), a suicide vector (pHS7) containing an antibiotic cassette was constructed to achieve the replacement, by homologous recombination, of the wild-type hemL gene in the chromosome by a modified form of the gene. Recombinant strains of Synechococcus 7942 obtained using pHS7-hemL ^GR6 were indistinguishable from Synechococcus 6301 GR6 in terms of the resistance of growth and of chlorophyll accumulation to high concentrations of gabaculine, while a wild-type recombinant produced using pHS7-hemL ^WT had retained its sensitivity. Southern hybridisation using gene probes for hemL, amp ^r and cm ^r confirmed that chromosomal integration of the plasmids had occurred in both WT and GR6 recombinants. Growth and chlorophyll accumulation in equivalent strains with the hemL gene containing either the deletion or the transition characteristic of Synechococcus 6301 GR6 were inhibited by 10 μM gabaculine. Consequently, resistance in vivo to high concentrations of this compound is dependent on both the changes in gene/enzyme structure. This investigation has established the effectiveness of the suicide vector pHS7 for studying the effect in vivo of specific changes in the hemL gene. It has also demonstrated that replacement of the wild-type gene by that from Synechococcus 6301 GR6 is sufficient to confer resistance in vivo to high concentrations of gabaculine. Received: 7 October 1996 / Accepted: 15 January 1997