Glucuronidase Gene Research Articles

Transient expression and stable integration of chimeric Gus gene in watermelon following electroporation

SUMMARYA novel technique for introducing foreign genes into intact plant organs has been demonstrated in watermelon (Citrullus lanatus L.) following electroporation of zygotic embryos and nodal buds using the chimeric -glucuronidase (gus) gene. DNA transfer was done under in vitro and in vivo conditions using plasmid constructs harbouring the gus reporter gene under the control of the constitutive promoter CaMV35S and nos terminator. The parameters for electroporation were optimized to study the influence of different plasmid constructs on the uptake, transient expression and stable integration of the transgene in the electroporated tissues. Maximum GUS expression was observed by electroporating the tissues with five electric pulses of 400 V cm–1 discharged from a 960 µF capacitor in the presence of 200 fig DNA ml–1. Microscopic observation of the thin sections of paraffin-embedded embryos clearly showed that the ß-glucuronidase expression was exclusively located inside the treated cells. Further, we confirmed the stable integration of chimeric gene in R1 progeny obtained from in vivo electroporated watermelon plants by dot blot and Southern blot analysis.

The 5′-Untranslated Region of the Tobacco Alcohol Dehydrogenase Gene Functions as an Effective Translational Enhancer in Plant

The alcohol dehydrogenase gene ( NtADH ) was previously isolated from tobacco BY2 suspension cultured cell. Expression of this gene was dramatically increased only during the early stationary phase, and the 5′-untranslated region (5′-UTR) was hypothesized to be involved in the stimulatory effect at the post-transcriptional level. In this paper, we investigated whether the NtADH 5′-UTR possesses the ability to positively enhance gene expression at the translational level. For easily estimating translational efficiency, we used β - glucuronidase ( GUS ) gene as a reporter and tobacco BY2 cell, Arabidopsis thaliana T87 cell, and rice Oryza sativa suspension cultured cells as host cells in a transient assay system. Compared with the control plasmid pBI221, insertion of the NtADH 5′-UTR enhanced GUS expression levels about 30- to 100-fold and 30- to 60-fold in transiently transformed BY2 and T87 cells, respectively. However, in transiently transformed O. sativa cells, expression was barely enhanced. In comparison with the 5′-UTR of tobacco mosaic virus (ω sequence), a known translational enhancer, the NtADH 5′-UTR enhanced translation to a similar level. Meanwhile, the translational efficiency was affected by the sequence context around the AUG initiation codon at the translational initiation step. Moreover, this NtADH 5′-UTR also worked in stable tobacco transformants. Therefore, it is expected that this 5′-UTR will serve as a powerful tool for enhancing foreign gene expression.

Microprojectile-mediated genetic transformation and regeneration of Chinese elm

A protocol for the genetic transformation and regeneration of Chinese elm ( Ulmus parvifolia Ja cq.) is reported. Cell suspension cultures were initiated from hypocotyls and were maintained on Murashige and Skoog (MS) basal semi-solid medium supplemented with varying concentrations of thiadiazuron (TDZ) and 1-naphthaleneacetic acid (NAA). Two week- old cell suspension cultures were bombarded with gold particles coated with plasmid harboring the Basta®resistance and â -glucuronidase genes. Elm cultures produced green calli within a month on selection media containing 20 mg L-1phosphinothrycin. These c alli were transferred to the same medium without the herbicide and were regenerated into shoots within 2 mo. The expression of uidA reporter gene in transformed elms was detected by histochemical analysis for â-glucuronidase activity. The presence of the Basta®resistance insert was demonstrated by polymerase chain reaction analysis. Key words:

조직배양을 이용한 Kentucky bluegrass(Poa pratensis L.)의 식물체 재분화 및 형질전화 조건의 검토

In this study, plant regeneration and Agrobacterium tumefaciens-mediated transformation Kentucky bluegrass(Poa pratensis L.) were evaluated. Three different types of calli were produced depending on the combinations of growth regulators. They were non-friable brown or gray-colored callus (type I), compact, friable and yellow or white-colored callus (typeII), and soft, watery translucent callus with differentiated structure (typeIII). The highest regenerable organogenic callus (typeII) was obtained on the medium containing 1mg/L, 2,4-D and 0.1mg/L BA. Additionally, the production of typeII calli increased significantly when AgNO<TEX>$_3$</TEX> was added to the callus induction and growth medium. The highest frequency of multiple shout formation from typeII callus was obtained on MS medium containing 1mg/L BA and 1mg/L Thidiazuron(TDZ). The organogenic calli(typeII) were inoculated with Agrobacterium tumefaciens strain EHA101 harboring the binary vector pIG121Hm with <TEX>$\beta$</TEX>-glucuronidase gene, and various factors were found to influence the transfer-DNA delivery efficiency. The highest transient GUS activity was observed on typeIIcallus. In the present work, we reported the first transient GUS activity of Kentucky bluegrass mediated by Agrobacterium tumefaciens. Our system may contribute to genetic improvement for breed-recalcirtrant grass species, Kentucky bluegrass.

Particle Inflow Gun을 이용한 벼 캘러스 내의 효율적 유전자 도입

We have developed a homemade particle inflow gun (PIG) system which has simple operation method, low price and high gene introduction efficiency into rice callus. Rice callus were inflowed with gold particles containing DNA of a plasmid, pIG121Hm, harboring intron GUS (-glucuronidase) gene, NPTII gene and HPT gene. For optimal GUS transient expression, the effects of parameters on DNA delivery efficiency of the PIG system was investigated by scoring transient GUS expression. The highest number of blue spots was observed at 16 mM of spemidine and 1.5 M of calcium chloride, respectively. And the amount of gold particles required for the best GUD expression was 2 mg. Optimum GUS transient expression was observed at target distance of 12 cm and helium pressure of 3.5 bar (50 psi). Gene introduction efficiency of the PIG system was observed almost similar to that of the Biolistic Gun (Bio-Rad Company). Since PIG system is simple to operate and one doesn`t need disposable accessaries, the PIG system can be easily applied to various replication experiments.

CIS-REGULATORY ELEMENTS FOR PISTIL SPECIFIC EXPRESSION IN S-RNASE PROMOTER REGION OF JAPANESE PEAR (PYRUS PYRIFOLIA NAKAI)

The pistil-specific and temporal expression of Japanese pear S-RNase gene is regulated by S-RNase promoter region. To identify the cis-regulatory elements on the S-RNase promoter of Japanese pear, we analysed GUS activities in transgenic tobacco plants carrying 9 sequential deletions of S3-RNase promoter fused to the -glucuronidase (GUS) gene. GUS activity was detected quantitatively in the pistils from the transgenic plants carrying the promoter deleted from -747 to -310. GUS activity was localized in the stigmatic secretory zone, and the transmitting tissues in the pistils but not in other tissues; pollen, anthers, petals, sepals, leaves and roots. GUS activities in the pistils increased steadily from 4 days before flowering. No transgenic pistils with deletions carrying -255 or shorter promoters displayed GUS activity, indicating that the critical cis-regulatory elements for pistil specific expression were present between -310 and -255 of S3-RNase promoter region. Furthermore, deletions from -552 to -356 led to the drastic decrease of GUS activity in the transgenic pistil, which assumes that enhancer-like element for the high level of expression in pistils are present within this region.

애기장대 형질전환 식물체의 세대경과에 따른 GUS유전자의 비활성화에 관한 연구

The effect of transgene inactivation in T2, T3 and F2 generations was analyzed in progeny seedlings which had been generated by Agrobacterium (LBA4404/pBI121)-mediated transformation in Arabidopsis thaliana. In a system which investigated in the expression of -glucuronidase(GUS)gene in kanamycin-resistant (ke )seedlings, GUS inactivated seedlings were observed in 5 of 12 tested lines of T2 generation and the frequency of GUS inactivation was approximately 2.3%. Lines with multi-copies of T-DNA exhibited severe GUS gene inactivation with the frequency of 5.8% in T2 generation. In T3 generation lines exhibited GUS gene inactivation with the frequency of 1.3%. In contrast, inactivation increased dramatically up to 12.6% in multi-copy T-DNA line. A similar phenomenon was also found in F2 progeny from a transgenic line which had been crossed with wild-type Arabidopsis plant, WS-O (GUS gene inactivation frequency 9.9%). These results indicate that the foreign gene introduced into the plant was inactivated progressively in its transmission during subsequent generations and the transgenic line with multi-copies of T-DNA tended to show more increased inactivation.n.

Detection and enumeration of coliforms in drinking water: current methods and emerging approaches

The coliform group has been used extensively as an indicator of water quality and has historically led to the public health protection concept. The aim of this review is to examine methods currently in use or which can be proposed for the monitoring of coliforms in drinking water. Actually, the need for more rapid, sensitive and specific tests is essential in the water industry. Routine and widely accepted techniques are discussed, as are methods which have emerged from recent research developments. Approved traditional methods for coliform detection include the multiple-tube fermentation (MTF) technique and the membrane filter (MF) technique using different specific media and incubation conditions. These methods have limitations, however, such as duration of incubation, antagonistic organism interference, lack of specificity and poor detection of slow-growing or viable but non-culturable (VBNC) microorganisms. Nowadays, the simple and inexpensive membrane filter technique is the most widely used method for routine enumeration of coliforms in drinking water. The detection of coliforms based on specific enzymatic activity has improved the sensitivity of these methods. The enzymes β- d galactosidase and β- d glucuronidase are widely used for the detection and enumeration of total coliforms and Escherichia coli, respectively. Many chromogenic and fluorogenic substrates exist for the specific detection of these enzymatic activities, and various commercial tests based on these substrates are available. Numerous comparisons have shown these tests may be a suitable alternative to the classical techniques. They are, however, more expensive, and the incubation time, even though reduced, remains too long for same-day results. More sophisticated analytical tools such as solid phase cytometry can be employed to decrease the time needed for the detection of bacterial enzymatic activities, with a low detection threshold. Detection of coliforms by molecular methods is also proposed, as these methods allow for very specific and rapid detection without the need for a cultivation step. Three molecular-based methods are evaluated here: the immunological, polymerase chain reaction (PCR) and in-situ hybridization (ISH) techniques. In the immunological approach, various antibodies against coliform bacteria have been produced, but the application of this technique often showed low antibody specificity. PCR can be used to detect coliform bacteria by means of signal amplification: DNA sequence coding for the lacZ gene (β-galactosidase gene) and the uidA gene (β- d glucuronidase gene) has been used to detect total coliforms and E. coli, respectively. However, quantification with PCR is still lacking in precision and necessitates extensive laboratory work. The FISH technique involves the use of oligonucleotide probes to detect complementary sequences inside specific cells. Oligonucleotide probes designed specifically for regions of the 16S RNA molecules of Enterobacteriaceae can be used for microbiological quality control of drinking water samples. FISH should be an interesting viable alternative to the conventional culture methods for the detection of coliforms in drinking water, as it provides quantitative data in a fairly short period of time (6 to 8 h), but still requires research effort. This review shows that even though many innovative bacterial detection methods have been developed, few have the potential for becoming a standardized method for the detection of coliforms in drinking water samples.

Isolation and Characterization of a New Virulence Gene (abvA) of Agrobacterium tumefaciens

A mutant (M-1) was isolated by transposon (Tn5) insertion mutagenesis of Agrobacterium tumefaciens (strain A-208, C58 chromosome, nopaline type T37 pTi, virulent). The M-1 mutant exhibited a complete avirulent phenotype on Kalanchoe daigremontiana leaf and Kalanchoe pinnata stem but a very attenuated virulent phenotype on root of Daucus carota. The mutant had one insertion of Tn5 in pTi. A wild-type target segment (2.3 kb) that included the site of Tn5 insertion in M-1 mutant was cloned. Introducing the 2.3 kb segment into M-1 complemented completely the avirulent phenotype, producing galls as big as strain A-208. The 2.3 kb segment was sequenced, identifying three open reading frames, ORF 1 (354 bp), ORF 2 (261 bp) and ORF 3 (801 bp) in the segment. A Tn5 was inserted between the third and fourth nucleotide of ORF 1 in M-1. The ORF 1 had no homology to any reported genes and thus was named the abvA gene. The ORF 3 had the high homology (identities 44%, positive 68%) to the gene of the sarcosine oxidase β subunit (accession no. sp/P40875). Introduction of the DNA segment (743 bp) containing the abvA gene and its promoter region into M-1 partially complemented the avirulent phenotype of the mutant, producing galls smaller than strain A-208. The abvA gene was distributed not only on nopaline-type pTi (T37) but also on octopine-type pTi (A6NC) and chromosome (C58) of A. tumefaciens. M-1, being avirulent on K. daigremontiana and K. pinnata, had a Tn5 insertion only in the abvA gene on pTi but not in the abvA gene on the chromosome, implying that the abvA gene on the chromosome in strain A-208 is not functional. A binary vector, pIG121-Hm, containing the β -glucuronidase (GUS) gene with an intron was introduced into M-1, which was then applied to leaves of K. daigremontiana to assay GUS activity for monitoring T-DNA transfer to the host nucleus. High GUS activity comparable to that in strain A-208 was detected in M-1 in spite of its inability to induce galls, suggesting that M-1 can transfer T-DNA into the host nucleus, but cannot integrate it into the chromosome.

Transient expression of a reporter gene changes significantly during somatic embryogenesis in alfalfa

Transient expression of the β -glucuronidase (GUS) gene driven by a 35S constitutive promoter was investigated during somatic embryo development in alfalfa (Medicago sativa L.) following micro-projectile bombardment. The level of gene expression varied with the different developmental stages and changed dramatically within the stages. During embryogenic callus induction (stage I), expression was low in explants freshly transferred to culture, but increased when cells started to divide and show competence for somatic embryogenesis. Gene expression, however, decreased when extensive callus formation began and reached the lowest observed level when callus was fully developed. Reporter gene expression was consistently low during embryogenic cell proliferation (stage II). In contrast, high levels of reporter gene expression were detected in non-embryogenic suspension cultures during cell proliferation. Extremely low reporter gene expression was detected in embryonal tissues and young embryos at the embryo development and maturation stage (stage III). Expression increased as the somatic embryos developed and reached the highest level when the embryos reached maturity. Transient expression levels were similar in excised petiole tissue across 10 alfalfa genotypes, which included both diploid and tetraploid genotypes. In general, reporter gene expression was found to be higher in more organised mature tissues and organs than in less-organised or young tissues. The developmental status of the cells and tissues appears to be an important factor in the degree of transient expression of the introduced gene. Changes of gene expression during embryogenesis and factors affecting gene expression are discussed. Key words: GUS expression, Medicago sativa, bombardment, forage

486 Expression of β-glucuronidase Gene in Aspen under Control of CaMV35S, Heat Shock and RolC Promoters

Transgenic hybrid aspens (Populus tremuloides × P. tremula) were produced by Agrobacterium-mediated transformation and confirmed by polymerase chain reaction. Three promoters (CaMV 35S, Heat shock, and Rol C) were used to drive transcription of chimeric genes -glucuronidase (GUS), npt-II, and rol B. Stem sections in ≈100 mm thick, leaf blades, and root tips of transgenic aspen were treated with X-Gluc solution for 2 to 12 h in a 37 °C incubator and fixed in a solution containing 5% formaldehyde, 5% acetic acid, and 20% ethanol (FAA) for 10 min. After washing with 50% ethanol twice and clearing with absolute ethanol until free of chlorophyll, the GUS expression (localization and intensity of blue staining) in leaf, stem, and root at different growth stages were evaluated and photographed under the light microscope. When CaMV35S and rol C were used as promoters, the GUS gene was expressed in all parts of mature stem except pith, with the strongest activity in phloem. The heat shock promoter gave rise to very strong expression only in epidermis and phloem. In the young stem, GUS activity was detected in epidermis, parenchyma, vascular cambium, and primary xylem in CaMV35S-GUS transformed aspen shoots. The rol C promoter produced GUS gene expression in all stem tissues. When the heat shock promoter was used, the GUS gene expressed in a more tissue-specific manner, especially in mature stems, with activity mainly in parenchyma. In young leaf tissues, the GUS activity was primarily located in veins and mesophyll. In the mature leaves, no blue staining was found in the main vein. In root tip, the GUS gene driven by CaMV35S and heat shock promoters were expressed in the columella, vascular, and root apical meristem with very strong expression in the root apical meristem. Aspen plants transformed by rol C-Gus construct showed less or no expression in the columella.

Simultaneous Detection and Differentiation of Escherichia coli Populations from Environmental Freshwaters by Means of Sequence Variations in a Fragment of the β- d -Glucuronidase Gene

A PCR-based denaturing-gradient gel electrophoresis (DGGE) approach was applied to a partial sequence of the beta-D-glucuronidase gene (uidA) for specific detection and differentiation of Escherichia coli populations according to their uidA sequence variations. Detection of sequence variations by PCR-DGGE and by PCR with direct sequencing correlated perfectly. Screening of 50 E. coli freshwater isolates and reference strains revealed 11 sequence types, showing nine polymorphic sites and an average number of pairwise differences between alleles of the uidA gene fragments (screened fragment length, 126 bp) of 2.3%. Among the analyzed strains a range of dominating to more rarely and/or uniquely observed E. coli sequence types was revealed. PCR-DGGE applied to fecally polluted river water samples simultaneously detected E. coli and generated a fingerprint of the mixed populations by separating the polymorphic uidA amplicons. No significant differences between non-cultivation-based and cultivation-based profiles were observed, suggesting that at least some members of all occurring sequence types could be cultivated. As E. coli is frequently used as a fecal indicator, this work is considered an important step towards a new, practical tool for the differentiation and tracing of fecal pollution in all kinds of waters.

Effects of rho-independent terminators on the expression of the prokaryoticβ-glucuronidase gene in tobacco

The threonine (thr) attenuator with a dyad symmetrical structure is from the regulatory region of the thr operon of Escherichia coli, and encodes RNA with a stem-and-loop structure followed by a stretch of uridine residues. The thr attenuator and its variants were subcloned into the region between the 35S promoter and β-glucuronidase (GUS) coding region, and the transient expression of GUS gene in tobacco protoplast was treated as a reporter for gene regulation in plants. Results from the 14 variants in the stem region of the thr attenuator indicated that both base pairing and sequence specificity in the G+C-rich region of the stem were important for the GUS expression, but 1 base mismatch in the A+U-rich region of stem did not affect the GUS expression in plants. Seven variants with nested deletion in the stretch of uridine residues were also analyzed, and the results suggested that the variants with the shorter uridine stretch produced more GUS protein than those with the longer stretch. Transgenic tobacco plants with the thr attenuator and its variants located between the 35S promoter and GUS coding region were also generated, and their steady state RNAs were hybridized with 2 radioactive antisense RNA probes which bound 5′ and 3′ of the thr hairpin, respectively. After the digestion of S1 nuclease, the amount of the nuclease-resistant transcript from the protection of the 5′ antisense RNA probe was much more than that from the protection of the 3′ probe in all tested variants. This result suggests that these dyad symmetries may affect transcription of plant RNA polymerase II.

Upstream regulatory regions from the maize Sh1 promoter confer tissue-specific expression of the , -glucuronidase gene in tomato

A promoter fusion (Sh35) combining upstream regulatory regions from the maize Sh1 promoter with a truncated 35S promoter, Δ9035 (–90 to +8) has been compared with the original Sh1 promoter for its capacity to promote expression of the β-glucuronidase (GUS) gene in stably transformed tomato plants. For both promoters, very faint GUS expression was detected in the vegetative tissues, and no expression was detected in the fruit pericarp tissues. However, in the seed, Sh1 promoted low GUS expression but Sh35 directed 25-fold higher GUS expression. For both constructs, the profile of GUS expression was similar to that of endogenous sucrose synthase activity, but maximal GUS activity was reached 15 days after the peak of sucrose synthase activity.

Characterization of CorR, a transcriptional activator which is required for biosynthesis of the phytotoxin coronatine.

Coronatine (COR) is a plasmid-encoded phytotoxin synthesized by several pathovars of phytopathogenic Pseudomonas syringae. The COR biosynthetic gene cluster in P. syringae pv. glycinea PG4180 is encoded by a 32-kb region which contains both the structural and regulatory genes needed for COR synthesis. The regulatory region contains three genes: corP, corS, and corR. corS is thought to function as a histidine protein kinase, whereas corP and corR show relatedness to response regulators of the two-component regulatory paradigm. In the present study, we investigated whether CorR is a positive activator of COR gene expression. We also studied whether CorR specifically binds the DNA region located upstream of cfl, a gene located at the 5' end of the gene cluster encoding coronafacic acid, the polyketide portion of COR. Complementation analysis with a corR mutant, PG4180.P2, and transcriptional fusions to a promoterless glucuronidase gene (uidA) indicated that CorR functions as a positive regulator of COR gene expression. Deletion analysis of the 5' end of the cfl upstream region was used to define the minimal region required for COR gene expression. A 360-bp DNA fragment located over 500 bp upstream from the cfl transcriptional start site was used in DNase I protection assays to define the specific bases bound by CorR. An area extending from -704 to -650 with respect to the cfl transcriptional start site was protected by DNase I footprinting, indicating a rather large area of protection. This area was also conserved in the promoter region for cmaA, which encodes a transcript containing genes for coronamic acid synthesis, another intermediate in the COR biosynthetic pathway. The results obtained in the current study suggest that both the coronafacic acid and the coronamic acid structural genes are controlled by CorR, a positive activator of COR gene expression.

Growth phase and temperature influence promoter activity, transcript abundance, and protein stability during biosynthesis of the Pseudomonas syringae phytotoxin coronatine.

The plant-pathogenic bacterium Pseudomonas syringae pv. glycinea PG4180.N9 synthesizes high levels of the polyketide phytotoxin coronatine (COR) at 18 degrees C, whereas no detectable toxin is produced at 28 degrees C. Previously, we reported that the temperature-sensitive activation of three promoters within the COR biosynthetic gene cluster might explain thermoregulation of COR biosynthesis. The present study was aimed at furthering our understanding of the transcriptional as well as the posttranslational effects of temperature on expression of cmaB, which encodes an enzyme involved in COR biosynthesis. Transcriptional fusions using a promoterless glucuronidase gene and Northern blot analyses were used to monitor promoter activities and transcript abundance for the cmaABT operon during bacterial growth at 18 and 28 degrees C. Promoter activity and transcription rates were maximal when cells were incubated at 18 degrees C and sampled at mid-logarithmic phase. Transcription declined moderately during the transition to stationary phase but remained higher at 18 C than at 28 degrees C. Western blot analysis indicated that CmaB accumulated in the late stationary phase of P. syringae cultures grown at 18 degrees C but not in cultures incubated at 28 degrees C. Temperature shift experiments indicated that CmaB stability was more pronounced at 18 degrees C than at 28 degrees C. Although temperature has a strong impact on transcription of COR biosynthetic genes, we propose that thermoregulation of protein stability might also control COR synthesis.

Particle mediated DNA delivery and transient expression of GUS gene in plated cells of rice

Culture conditions for a fine dispersion of plated cells of Oryza sativa L. cv. IR 20, have been worked out. These plated cells developed microcalli containing large number of somatic embryos and subsequently plantlets. By using single cells and clusters of 2 - 4 cells, an efficient DNA-delivery by microprojectile bombardment into cells and its transient expression were assessed by employing a plasmid construct containing &beta;-glucuronidase gene.

Expression and analysis of coronafacate ligase, a thermoregulated gene required for production of the phytotoxin coronatine in Pseudomonas syringae

Coronafacic acid, the polyketide component of the phytotoxin coronatine, is activated and coupled to coronamic acid via amide bond formation, a biosynthetic step presumably catalyzed by the corona facate ligase ( cfl) gene product. In the present study, cfl was fused to the carboxy terminus of malE, which encodes the maltose-binding protein (MBP), and overexpressed in Escherichia coli. Immunoblot analysis indicated that Cfl contained an ATP-binding region, a motif conserved in enzymes which activate their substrates by adenylation. MBP-Cfl was overproduced and purified from Pseudomonas syringae and the protein fusion was used to generate antisera. Anti-MBP-Cfl antibodies and a transcriptional fusion of the cfl promoter to a promoterless glucuronidase gene were used to follow the temporal expression of coronafacate ligase. The results indicated that transcription of cfl is temperature-sensitive. Furthermore, a nonpolar mutation in cfl suggested that the gene may have a role in coronafacic acid biosynthesis.

Characterization of the alginate biosynthetic gene cluster in Pseudomonas syringae pv. syringae.

Alginate, a copolymer of D-mannuronic acid and L-guluronic acid, is produced by a variety of pseudomonads, including Pseudomonas syringae. Alginate biosynthesis has been most extensively studied in P. aeruginosa, and a number of structural and regulatory genes from this species have been cloned and characterized. In the present study, an alginate-defective (Alg-) mutant of P. syringae pv. syringae FF5 was shown to contain a Tn5 insertion in algL, a gene encoding alginate lyase. A cosmid clone designated pSK2 restored alginate production to the algL mutant and was shown to contain homologs of algD, alg8, alg44, algG, algX (alg60), algL, algF, and algA. The order and arrangement of the structural gene cluster were virtually identical to those previously described for P. aeruginosa. Complementation analyses, however, indicated that the structural gene clusters in P. aeruginosa and P. syringae were not functionally interchangeable when expressed from their native promoters. A region upstream of the algD gene in P. syringae pv. syringae was shown to activate the transcription of a promoterless glucuronidase (uidA) gene and indicated that transcription initiated upstream of algD as described for P. aeruginosa. Transcription of the algD promoter from P. syringae FF5 was significantly higher at 32 degrees C than at 18 or 26 degrees C and was stimulated when copper sulfate or sodium chloride was added to the medium. Alginate gene expression was also stimulated by the addition of the nonionic solute sorbitol, indicating that osmolarity is a signal for algD expression in P. syringae FF5.

Transient b-glucuronidase activity after infiltration of Arabidopsis thaliana by Agrobacterium tumefaciens

Transient expression of the &beta;-glucuronidase (GUS) gene introduced into Arabidopsis thaliana intact plants by T-DNA after vacuum infiltration of Agrobacterium tumefaciens was followed. The first incidence of GUS activity was found 2 - 3 d after treatment and a peak of activity one week after treatment in both A. thaliana races, Columbia and C24. GUS activity was sharply increased by cultivation of Arabidopsis plants at elevated temperature (29 °C) compared to cultivation at 25 °C. The density of inocula also influenced the GUS activity.