Rat liver beta-glucuronidase. cDNA cloning, sequence comparisons and expression of a chimeric protein in COS cells.

A cDNA for rat liver beta-glucuronidase was isolated, its sequence determined and its expression after transfection into COS cells studied. The deduced amino acid sequence of the rat liver clone showed 77% homology with that from the cDNA for human placental beta-glucuronidase and 47% homology with that deduced from the cDNA for Escherichia coli beta-glucuronidase. Several differences were found between the cDNA from rat liver and that previously reported from rat preputial gland. Only one change leads to an amino acid difference in the mature enzyme. A chimeric clone was constructed by using a fragment encoding the first 18 amino acid residues of the signal sequence from the human placental cDNA clone and a fragment from the rat clone encoding four amino acid residues of the signal sequence, all 626 amino acid residues of the mature rat enzyme, and all of the 3' untranslated region. After transfection into COS cells the chimeric clone expressed beta-glucuronidase activity that was specifically immunoprecipitated by antibody to rat beta-glucuronidase. The Mr value of 76,000 of the expressed gene product was characteristic of the glycosylated rat enzyme. It was proteolytically processed in COS cells to Mr 75,000 6 h after metabolic labelling. At least 50% of the expressed enzyme was secreted at 60 h post-transfection, but the secreted enzyme did not undergo proteolytic processing. These results provide evidence that the partial cDNA isolated from a rat liver library contains the complete coding sequence for the mature rat liver enzyme and that the chimeric signal sequence allows normal biosynthesis and processing of the transfected rat liver enzyme in COS cells.     

Cell position and light influence C4 versus C3 patterns of photosynthetic gene expression in maize.

C4 plants such as maize partition photosynthetic activities in two morphologically distinct cell types, bundle sheath (BS) and mesophyll (M), which lie as concentric layers around veins. We show that both light and cell position relative to veins influence C4 photosynthetic gene expression. A pattern of gene expression characteristic of C3 plants [ribulose bisphosphate carboxylase (RuBPCase) and light-harvesting chlorophyll a/b binding protein in all photosynthetic cells] is observed in leaf-like organs such as husk leaves, which are sparsely vascularized. This pattern of gene expression reflects direct fixation of CO2 in the C3 photosynthetic pathway, as determined by O2 inhibition assays. Light induces a switch from C3-type to C4-type gene expression patterns in all leaves, primarily in cells that are close to a vein. We propose that light causes repression of RuBPCase expression in M cells, by a mechanism associated with the vascular system, and that this is an essential step in the induction of C4 photosynthesis.     

The first and fourth upstream open reading frames in GCN4 mRNA have similar initiation efficiencies but respond differently in translational control to change in length and sequence.

The third and fourth AUG codons in GCN4 mRNA efficiently repress translation of the GCN4-coding sequences under normal growth conditions. The first AUG codon is approximately 30-fold less inhibitory and is required under amino acid starvation conditions to override the repressing effects of AUG codons 3 and 4. lacZ fusions constructed to functional, elongated versions of the first and fourth upstream open reading frames (URFs) were used to show that AUG codons 1 and 4 function similarly as efficient translational start sites in vivo, raising the possibility that steps following initiation distinguish the regulatory properties of URFs 1 and 4. In accord with this idea, we observed different consequences of changing the length and termination site of URF1 versus changing those of URFs 3 and 4. The latter were lengthened considerably, with little or no effect on regulation. In fact, the function of URFs 3 and 4 was partially reconstituted with a completely heterologous URF. By contrast, certain mutations that lengthen URF1 impaired its positive regulatory function nearly as much as removing its AUG codon did. The same mutations also made URF1 a much more inhibitory element when it was present alone in the mRNA leader. These results strongly suggest that URFs 1 and 4 both function in regulation as translated coding sequences. To account for the phenotypes of the URF1 mutations, we suggest the most ribosomes normally translate URF1 and that the mutations reduce the number of ribosomes that are able to complete URF1 translation and resume scanning downstream. This effect would impair URF1 positive regulatory function if ribosomes must first translate URF1 in order to overcome the strong translational block at the 3'-proximal URFs. Because URF1-lacZ fusions were translated at the same rate under repressing and derepressing conditions, it appears that modulating initiation at URF1 is not the means that is used to restrict the regulatory consequences of URF1 translation to starvation conditions.     

Meclofenamate blocks the pulmonary arterial vasopressor effects of leukotriene B4 in awake sheep

This study examined the hemodynamic effects of leukotriene B4 (LTB4) in chronically instrumented awake sheep, and the role of cyclooxygenase products in the sheep's response to LTB4. LTB4 (25 micrograms) was given as a bolus into the pulmonary artery. Six sheep were studied with LTB4, both before and after pretreatment with meclofenamate (5 mg/kg load, 3 mg/kg/hr maintenance infusion). LTB4 alone caused a rapid rise in pulmonary arterial pressure from 15 +/- 1 to 42 +/- 11 cm H2O. LTB4 had no effect on pulmonary arterial pressure following pretreatment with meclofenamate. LTB4 alone caused an increase in serum thromboxane B2 (TxB2) from 130 +/- 35 to 320 +/- 17 pg/ml 3 minutes after dosing but did not increase TxB2 following pre-treatment with meclofenamate. LTB4 caused a slight decrease in mean systemic arterial pressure and a transient fall in circulating white blood cells, both of which were unaffected by meclofenamate pre-treatment. The vasoactive effects of LTB4 in the pulmonary circulation appear to be mediated indirectly through the production of cyclooxygenase metabolites of arachidonic acid.     

Sequence comparison with concave weighting functions

We consider efficient methods for computing a difference metric between two sequences of symbols, where the cost of an operation to insert or delete a block of symbols is a concave function of the block's length. Alternatively, sequences can be optimally aligned when gap penalties are a concave function of the gap length. Two algorithms based on the ‘candidate list paradigm’ first used by Waterman (1984) are presented. The first computes significantly more parsimonious candidate lists than Waterman's method. The second method refines the first to the point of guaranteeingO(N ² lgN) worst-case time complexity, and under certain conditionsO(N ²). Experimental data show how various properties of the comparison problem affect the methods' relative performance. A number of extensions are discussed, among them a technique for constructing optimal alignments inO(N) space in expectation. This variation gives a practical method for comparing long amino sequences on a small computer. This work was supported in part by NSF Grant DCR-8511455.     

Dissimilar plasmids isolated from Pseudomonas diminuta MG and a Flavobacterium sp. (ATCC 27551) contain identical opd genes

The opd (organophosphate-degrading) gene derived from a 43-kilobase-pair plasmid (pSM55) of a Flavobacterium sp. (ATCC 27551) has a sequence identical to that of the plasmid-borne gene of Pseudomonas diminuta. Hybridization studies with DNA fragments obtained by restriction endonuclease digestion of plasmid DNAs demonstrated that the identical opd sequences were encoded on dissimilar plasmids from the two sources.     

Conjugal transfer of R68.45 and FP5 between Pseudomonas aeruginosa strains in a freshwater environment

Recent concern over the release of genetically engineered organisms has resulted in a need for information about the potential for gene transfer in the environment. In this study, the conjugal transfer in Pseudomonas aeruginosa of the plasmids R68.45 and FP5 was demonstrated in the freshwater environment of Fort Loudoun Resevoir, Knoxville, Tenn. When genetically well defined plasmid donor and recipient strains were introduced into test chambers suspended in Fort Loudoun Lake, transfer of both plasmids was observed. Conjugation occurred in both the presence and absence of the natural microbial community. The number of transconjugants recovered was lower when the natural community was present. Transfer of the broad-host-range plasmid R68.45 to organisms other than the introduced recipient was not observed in these chambers but was observed in laboratory simulations when an organism isolated from lakewater was used as the recipient strain. Although the plasmids transferred in laboratory studies were genetically and physically stable, a significant number of transconjugants recovered from the field trials contained deletions and other genetic rearrangements, suggesting that factors which increase gene instability are operating in the environment. The potential for conjugal transfer of genetic material must be considered in evaluating the release of any genetically engineered microorganism into a freshwater environment.     

Expression and Secretion of a Cellulomonas fimi Exoglucanase in Saccharomyces cerevisiae

We used the yeast MEL1 gene for secreted alpha-galactosidase to construct cartridges for the regulated expression of foreign proteins from Saccharomyces cerevisiae. The gene for a Cellulomonas fimi beta-1,4-exoglucanase was inserted into one cartridge to create a fusion of the alpha-galactosidase signal peptide to the exoglucanase. Yeast transformed with plasmids containing this construction produced active extracellular exoglucanase when grown under conditions appropriate to MEL1 promoter function. The cells also produced active intracellular enzyme. The secreted exoglucanase was N-glycosylated and was produced continuously during culture growth. It hydrolyzed xylan, carboxymethyl cellulose, 4-methylumbelliferyl-beta-d-cellobiose, and p-nitrophenyl-beta-d-cellobiose. A comparison of the recombinant S. cerevisiae enzyme with the native C. fimi enzyme showed the yeast version to have an identical K(m) and pH optimum but to be more thermostable.     

Selective solubilization of xylan in pulp using a purified xylanase fromTrichoderma harzianum

Summary The specificity of action of a cellulase-free xylanase preparation on pulp fibers was revealed by the composition of the solubilized products after enzyme treatment. The neutral carbohydrates released by the treatment of two hardwood kraft pulps were composed exclusively of xylooligomers. A similar treatment of Solka Floc showed no detrimental effect on the degree of polymerization of the cellulose fibers, as determined by size exclusion chromatographic analysis.     

Morphological characterization of cultured bovine aortic endothelial cells and the effects of atriopeptin II and sodium nitroprusside on cellular and extracellular accumulation of cyclic GMP

Primary, first and second passaged endothelial cells from bovine aorta were grown in plastic culture dishes or on glass coverslips. The cells were characterized by their monolayer cobblestone appearance at confluence, their immunofluorescent staining for factor VIII-related antigen, their specific uptake of low density lipoprotein and by their ultrastructure. Following stimulation of the cells by atriopeptin II or sodium nitroprusside, both cellular and extracellular cyclic GMP levels were measured. Cellular cyclic GMP content was increased greatly by atriopeptin II in a time-dependent manner while sodium nitroprusside was essentially without effect. Increases in tissue cyclic GMP levels were associated with a time-dependent accumulation of the nucleotide in the extracellular compartment. Zaprinast, a specific inhibitor of cyclic GMP phosphodiesterases, did not significantly affect either basal or atriopeptin II-stimulated increases in cyclic GMP content, nor extracellular accumulation of the nucleotide. It is concluded that the cyclic GMP content of endothelial cells is not solely dependent on degradation by phosphodiesterases but also involves release of cyclic GMP into the extracellular compartment.