Design of a degenerate primer pair to target a bacterial functional community: The hppd bacterial gene coding for the enzyme targeted by herbicides, a study case

ABSTRACT d-Galacturonic acid can be obtained by hydrolyzing pectin, which is an abundant and low value raw material. By means of metabolic engineering, we constructed fungal strains for the conversion of d-galacturonate to meso-galactarate (mucate). Galactarate has applications in food, cosmetics, and pharmaceuticals and as a platform chemical. In fungi d-galacturonate is catabolized through a reductive pathway with a d-galacturonate reductase as the first enzyme. Deleting the corresponding gene in the fungi Hypocrea jecorina and Aspergillus niger resulted in strains unable to grow on d-galacturonate. The genes of the pathway for d-galacturonate catabolism were upregulated in the presence of d-galacturonate in A. niger, even when the gene for d-galacturonate reductase was deleted, indicating that d-galacturonate itself is an inducer for the pathway. A bacterial gene coding for a d-galacturonate dehydrogenase catalyzing the NAD-dependent oxidation of d-galacturonate to galactarate was introduced to both strains with disrupted d-galacturonate catabolism. Both strains converted d-galacturonate to galactarate. The resulting H. jecorina strain produced galactarate at high yield. The A. niger strain regained the ability to grow on d-galacturonate when the d-galacturonate dehydrogenase was introduced, suggesting that it has a pathway for galactarate catabolism.

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Ontogenic expression of a CyI actin fusion gene injected into sea urchin eggs

Development ◽

10.1242/dev.101.3.437 ◽

1987 ◽

Vol 101 (3) ◽

pp. 437-447

Author(s):

K.S. Katula ◽

B.R. Hough-Evans ◽

R.J. Britten ◽

E.H. Davidson

Keyword(s):

Sea Urchin ◽

Fusion Gene ◽

Transcription Unit ◽

Upstream Region ◽

Upstream Sequence ◽

Flanking Sequence ◽

Bacterial Gene ◽

Fusion Construct ◽

Gene Coding ◽

Sea Urchin Egg

The 5′ terminus of the CyI actin gene transcription unit of Strongylocentrotus purpuratus was located by primer extension and other procedures, and the flanking upstream region was partially sequenced and mapped. A fusion gene was constructed containing about 2.5 kb of 5′ flanking sequence, the transcribed leader sequence, and the first few codons of the CyI gene ligated to the bacterial gene coding for chloramphenicol acetyl transferase (CAT). This was micro-injected into the cytoplasm of S. purpuratus eggs, and CAT enzyme activity was measured at various stages of embryonic development. CAT synthesis was activated between 10 and 14 h postfertilization, the same time at which newly synthesized transcripts of the endogenous CyI gene first appear. The exogenous CyI.CAT fusion DNA replicated actively during cleavage, as observed previously for other DNAs injected into sea urchin egg cytoplasm. Thus the absence of CAT activity prior to 10 h postfertilization could not be due to insufficient CyI.CAT genes. The amounts of CAT enzyme produced by embryos bearing CyI.CAT deletions that lack various regions of the CyI sequence were measured. As little as 254 nucleotides of upstream CyI sequence suffice for correct temporal activation of the fusion construct, although the level of CAT enzyme produced in embryos bearing any deletion retaining less than 850 nucleotides of upstream sequence was significantly lowered compared to controls bearing the complete CyI.CAT fusion construct.

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Expression in Plants of a Bacterial Gene Coding for Glyphosate Resistance

Weed Science ◽

10.1017/s0043174500060999 ◽

1987 ◽

Vol 35 (S1) ◽

pp. 19-23 ◽

Cited By ~ 17

Author(s):

Gregory A. Thompson ◽

William R. Hiatt ◽

Daniel Facciotti ◽

David M. Stalker ◽

Luca Comai

Keyword(s):

Single Amino Acid ◽

Kinetic Properties ◽

Epsp Synthase ◽

Bacterial Gene ◽

Gene Coding ◽

Bacterial Enzyme ◽

Genetically Engineer ◽

Single Amino Acid Change ◽

Low Sensitivity ◽

Dna Vectors

The target site of glyphosate [N-(phosphonomethyl)glycine] inhibition in plants and bacteria is 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase. Our strategy for developing glyphosate-resistant crops has been to genetically engineer plants with a gene that codes for EPSP synthase with low sensitivity in glyphosate. We cloned such a gene from thearoAlocus of a glyphosate-resistant mutagenized strain ofSalmonella typhimurium.The enzyme encoded by this gene has a single amino acid change resulting in lower affinity for glyphosate and higher affinity for substrates than either plant or wild-type bacterial counterpart. A chimaeric gene containing the mutantaroAgene behind the octopine synthase promoter was constructed and integrated intoAgrobacteriumT-DNA vectors. Analysis of gall tissue fromBrassica campestrisL. (turnip rape) infected withA. tumefaciensK12 containing this chimaera showed mRNA and protein expressed from the bacterial gene; 50% of the total EPSP synthase activity present had kinetic properties of the mutant bacterial enzyme. Tobacco (Nicotiana tabacumL. ‘Xanthi′) plants have been regenerated from cocultivation withA. rhizogenescontaining the same construct; analysis indicates expression of the gene and enhanced tolerance to glyphosate.

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EXPRESSION IN PLANTS OF A BACTERIAL GENE CODING FOR GLYPHOSATE RESISTANCE

Biotechnology in Plant Science ◽

10.1016/b978-0-12-775310-2.50032-6 ◽

1985 ◽

pp. 329-338

Author(s):

Luca Comai ◽

Daniel Facciotti ◽

David M. Stalker ◽

Gregory A. Thompson ◽

William R. Hiatt

Keyword(s):

Glyphosate Resistance ◽

Bacterial Gene ◽

Gene Coding

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Sonic Hedgehog is a member of the Hh/DD-peptidase family that spans the eukaryotic and bacterial domains of life

10.1101/276295 ◽

2018 ◽

Author(s):

Henk Roelink

Keyword(s):

Catalytic Activity ◽

Sonic Hedgehog ◽

Birth Defect ◽

Bacterial Gene ◽

Bacterial Proteins ◽

Gene Coding ◽

Domains Of Life ◽

Hh Signaling ◽

Congenital Birth Defect ◽

Stable Forms

AbstractSonic Hedgehog (Shh) coordinates Zn2+ in a manner that resembles that of peptidases. The ability of Shh to undergo autoproteolytic processing is impaired in mutants that affect the Zn2+ coordination, while mutating residues essential for catalytic activity results in more stable forms of Shh. The residues involved in Zn2+ coordination in Shh are found mutated in some individuals with the congenital birth defect holoprosencephaly, demonstrating their importance in development. Highly conserved Shh domains are found a parts of some bacterial proteins that are members of the larger family of DD-peptidases, supporting the notion that Shh acts as a peptidase. Whereas this Hh/DD-peptidase motif is present in Hedgehog (Hh) proteins of nearly all animals, it is not present in Drosophila Hh, indicating that Hh signaling in fruit flies is derived, and perhaps not a good model for vertebrate Shh signaling. Sequence analysis of Hh proteins and their possible evolutionary precursors suggest that the evolution of modern Hh might have involved horizontal transfer of a bacterial gene coding of a Hh/DD-peptidase into a Cnidarian ancestor, recombining to give rise to modern Hh.

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Banded aggregates containing fibrillin are present in the cartilage matrix adjacent to chondrocytes in individuals affected with scoliosis

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100124860 ◽

1992 ◽

Vol 50 (1) ◽

pp. 892-893

Author(s):

Douglas R. Keene ◽

Magaret Fairhurst ◽

Catherine C. Ridgway ◽

Lynn Y. Sakai

Keyword(s):

Connective Tissue ◽

Marfan Syndrome ◽

Cross Section ◽

Immunoelectron Microscopy ◽

Cartilage Matrix ◽

Negative Stain ◽

Gene Coding ◽

Rotary Shadowing

Matrix microfibrils are present in the connective tissue matrices of all tissues. Following standard TEM processing, they appear in cross section as cylindrical fibrils 8-10 nm in diameter, often associated with amorphous elastin. They are also seen in the absence of amorphous elastin, for example in the shallow papillary layer of skin, and also in cartilage matrix (Figure 1). Negative stain and rotary shadowing studies suggest that microfibrils are composed of laterally associated globular structures connected by fine filamentous strands (“ beaded strings”), and that they are extendable. Immunoelectron microscopy has demonstrated that fibrillin, a 350 Kd glycoprotein, is distributed along all microfibrils with a relaxed periodicity of about 54 nm The gene coding for fibrillin has recently been identified and is defective in the Marfan syndrome.

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