Critical nature of a specific uridine O2-carbonyl for cleavage by the hammerhead ribozyme.

Three modified hammerhead ribozyme/substrate complexes have been prepared in which individual uridine O2-carbonyls have been eliminated. The modified complexes were chemically synthesized with the substitution of a single 2-pyridone (2P) base analogue for residues U4, U7, and U16.1. Steady-state kinetic analyses indicate that the cleavage efficiencies for the U7 and U16.1 complexes were not significantly reduced relative to the native complex as measured by kcat/KM. The cleavage efficiency for the 2P4 complex, with the analogue present within the uridine loop, was reduced by greater than 2 orders of magnitude. This significant reduction in catalytic efficiency was due primarily to a decrease in kcat. The pH vs cleavage rate profile suggests that the O2-carbonyl of the U4 residue of the hammerhead complex is critical for transition state stabilization and efficient cleavage activity. The results of a Mg2+ rescue assay do not implicate the O2-carbonyl of U4 in an interaction with a divalent metal ion. In addition, the results of a ribozyme folding assay suggest that the presence of the 2P4 within the uridine loop does not alter the folding pathway (relative to the native sequence) both in the absence and in the presence of Mg2+. The O2-carbonyl of U4 appears oriented toward the interior of the catalytic pocket where it may be involved in a critical hydrogen bonding interaction necessary for transition state stabilization.

Phytochelatin synthase genes from Arabidopsis and the yeast Schizosaccharomyces pombe.

Phytochelatins (PCs), a family of heavy metal-inducible peptides important in the detoxification of heavy metals, have been identified in plants and some microorganisms, including Schizosaccharomyces pombe, but not in animals. PCs are synthesized enzymatically from glutathione (GSH) by PC synthase in the presence of heavy metal ions. In Arabidopsis, the CAD1 gene, identified by using Cd-sensitive, PC-deficient cad1 mutants, has been proposed to encode PC synthase. Using a positional cloning strategy, we have isolated the CAD1 gene. Database searches identified a homologous gene in S. pombe, and a mutant with a targeted deletion of this gene was also Cd sensitive and PC deficient. Extracts of Escherichia coli cells expressing a CAD1 cDNA or the S. pombe gene catalyzing GSH-dependent, heavy metal-activated synthesis of PCs in vitro demonstrated that both genes encode PC synthase activity. Both enzymes were activated by a range of metal ions. In contrast, reverse transcription-polymerase chain reaction experiments showed that expression of the CAD1 mRNA is not influenced by the presence of Cd. A comparison of the two predicted amino acid sequences revealed a highly conserved N-terminal region, which is presumed to be the catalytic domain, and a variable C-terminal region containing multiple Cys residues, which is proposed to be involved in activation of the enzyme by metal ions. Interestingly, a similar gene was identified in the nematode, Caenorhabditis elegans, suggesting that PCs may also be expressed in some animal species.

Gait improvement after carotid endarterectomy.

A prospective study was performed to investigate whether carotid endarterectomy was accompanied by changes in the cerebral circulation and gait of patients with gait disturbance caused by cerebral ischemia from an internal carotid artery flow lesion. Gait analysis was performed pre-and postoperatively in 16 patients who had a gait disturbance from April 1997 to May 1998. A correlation between change of volume flow measured by duplex sonography, a change in cerebral perfusion (CP) by single photon emission computed tomography (SPECT), the status of collateral circulation by cerebral angiogram or magnetic resonance angiogram, and improvement of gait disturbance was analyzed. Of 16 patients with gait disturbance, 14 (87.5%) showed improvement in various gait analysis parameters. Statistical significance was noted in cadence, speed, stride length, step time, and knee range of motion (P<05). Dramatic gait improvement was observed in 8 patients who had markedly increased Doppler volume flow after carotid endarterectomy. Among those 8 patients, 7 had a significant increase of CP as measured by SPECT and an incomplete circle of Willis. In conclusion, the most significant gait improvement was noted in patients who had severely decreased preoperative volume flow, an incomplete circle of Willis, and significantly increased postoperative CP as measured by SPECT.

Efficient selection and regeneration of creeping bentgrass transformants following particle bombardment.

We have established an efficient genetic transformation system for creeping bentgrass (Agrostis palustris Huds.) using particle bombardment. The transformation was performed using the plasmid pZO1052 which contains the reporter ß-glucuronidase (uidA) gene and the selectable marker hygromycin phosphotransferase (hph) gene. Transformed calli and plants were obtained via particle bombardment followed by selection of transformants on medium containing 200 mg/l of hygromycin. An average of 4.6 resistant colonies per bombardment were obtained. Southern analysis confirmed the integration of foreign genes in 19 of 21 putative transformants, indicating that selection by hygromycin was highly effective.

Adventitious bursae in below knee amputees. Case reports and a review of the literature.

Adventitious bursae are known to occur from repeated friction and pressure but rarely have been reported in adult amputees. Four cases of adventitious bursae are described, which were noted during the course of a follow-up study of below knee amputees. Two bursae resolved with modification of the socket or refabrication. One developed bursitis and drainage and a fourth persisted in a patient with systemic lupus erythematosus.

Transgenic turf-type tall fescue (Festuca amndinacea Schreb.) plants regenerated from protoplasts.

To improve turfgrasses using genetic engineering, we have developed a transformation system in turf-type tall fescue, one of the most important turfgrass species. Embryogenic cell cultures were established after callus induction from embryos of mature seed. The agarose-bead method with nurse cells was used to culture protoplasts and plants were regenerated from protoplasts of tall fescue cultured cells. To develop transgenic tall fescue plants, the hygromycin resistance gene and the ß-glucuronidase gene were introduced into the tall fescue protoplasts by electroporation. A high concentration (200 mg/l) of hygromycin was required to select transformed cells because of the high level of endogenous resistance to the antibiotic in tall fescue. Most of the transformed cells exhibited GUS activity and several plants were regenerated from these cells. The presence of introduced genes was confirmed by Southern blot hybridization of PCR amplified DNA from transgenic plants.

Nopaline synthase promoter is wound inducible and auxin inducible.

The activity of the nopaline synthase (nos) promoter is differentially regulated in several plant organs. In this article we demonstrate that the nos promoter is wound inducible in both vegetative and reproductive organs. The induction of the nos promoter was observed in leaves, stems, cotyledons, and various reproductive organs, suggesting that the response is not organ specific. The wound response was further enhanced by addition of auxins. Other growth substances had no effect on the wound-inducible nos promoter activity. Deletion analysis of the nos promoter indicated that the 10-base pair (GCACATACGT) Z element located between -123 and -114 or an element overlapping with this sequence is essential for the wound and auxin responses.

Cis-acting regulatory elements controlling temporal and organ-specific activity of nopaline synthase promoter.

Regulatory elements controlling temporal and organ-specific expression of the nopaline (nos) gene were identified by analyzing deletion mutants of the promoter. As observed in cultured cells, the TATA box element was required for efficient promoter function and the 29 bp upstream promoter region between -130 and -101 was necessary for the nos promoter activity in various vegetative organs. This 29 bp region includes ten nucleotides of a potential Z-DNA-forming sequence (Z element) and eight nucleotides of a repeated element (b element). Duplication of b elements significantly enhanced the promoter strength, revealing the importance of the element in all plant organs. Unlike the results in the cultured cells, however, deletion of the b element or CCAAT box region completely inactivated the promoter function in regenerated organs. Therefore, it appears that transcription initiation is more tightly controlled in differentiated plant cells than in cultured cells.

Organ-specific and developmental regulation of the nopaline synthase promoter in transgenic tobacco plants.

Control regions of the nopaline synthase (nos) gene have been widely used to express foreign genes in plants since the promoter is active in a wide variety of plant tissues. We report here the characteristics of the nos promoter activity in transgenic tobacco (Nicotiana tabacum) plants at various developmental stages. The promoter was highly active in the lower parts of a plant and gradually decreased in the upper parts. This vertical gradient was maintained throughout plant growth until the flowering stage when the overall promoter strength decreased significantly in the vegetative organs. However, in various flower organs, the nos promoter activities increased dramatically. Higher activity was observed in calyx, corolla, and stamens although the maximum promoter activity in each organ was found at different stages of flower development. The promoter activity in pistils was low and gradually increased in the ovaries after anthesis. In developing fruits, the nos promoter activity was strongly induced during the mid-stage of embryogenesis. These results indicate that the expression of the nos promoter is developmentally regulated and organ specific in transgenic tobacco plants.

Identification of upstream regulatory elements involved in the developmental expression of the Arabidopsis thaliana cab1 gene.

We studied cis regulatory elements controlling the light-dependent organ-specific expression of Arabidopsis thaliana chlorophyll a/b binding protein gene (cab1) by stably transforming tobacco plants using a tumor-inducing (Ti) plasmid vector system. The results from the 5' and internal deletion analyses indicate that there are at least three cis-acting elements that are involved in the light-dependent developmental expression of cab1 gene. Two such elements are located at the immediate upstream regulatory region and the other element is located at the further upstream region. The 1120-base-pair (bp) DNA fragment containing the immediate and far upstream region can confer light-inducible organ specificity on the truncated nos promoter. However, deletion of the 39-bp DNA fragment at the immediate upstream regulatory region from this hybrid promoter resulted in a nonfunctional promoter, revealing that the 39-bp region is important for the cab promoter specificity. Further analyses of this region suggest that a potential Z-DNA-forming sequence (ATACGTGT) is involved in light-dependent developmental expression of the cab1 gene. Two additional Z-DNA-forming sequences (ACACATAT) that are inverted repeats of this sequence are also found in the upstream region where the additional regulatory elements are expected.

Identification of an essential upstream element in the nopaline synthase promoter by stable and transient assays.

We studied the fine structure of the nopaline synthase (nos) promoter, which is active constitutively in a wide range of plant tissues, by both transient and stable transformation expression analyses. 3' and 5' deletion fragments were linked to form a set of internal deletion and duplication mutants that scanned the nos promoter. These mutated promoters were linked to the gene for the marker chloramphenicol acetyltransferase (CATase) as a means to readily assay promoter strength. The stable transformation analysis revealed the functional importance of an extended CCAAT box region (-97 to -63). Deletion of an upstream region (-112 to -101) containing an octameric repeated element resulted in a reduction in promoter strength by a factor of 30. A further deletion (-119 to -101) disrupted a potential Z-DNA-forming element as well, totally eliminating promoter function. Thus, a 19-base deletion across a repeated octamer and a potential Z-DNA-forming element identifies an essential upstream activator in the nos promoter. Duplication of the upstream element tripled promoter activity. Electroporation-mediated transient analysis was unable to distinguish downstream promoter elements. However, the upstream element behaved similarly in both assays in that deletion of the entire upstream element resulted in no promoter activity and that duplication of the element significantly enhanced the promoter strength.

I-cell disease and its rehabilitation: case study.

I-cell disease is a rare inborn error of mucolipid metabolism that is characterized by generalized hypotonia, thick and tight skin, restriction of joint motion, coarse facial features, bony deformities, and an inability to stand or walk. A case was treated with gentle stretching, neurodevelopmental therapy, and strengthening exercises of both hip and knee extensor muscles. After this treatment the patient was able to ambulate with moderate support using bilateral long leg braces.