Electrocontact microdisplacement testing subsystem

Zagursky, V.; Zibinch, D.

doi:10.1109/autest.1999.800404

Cited by 1 publication

(1 citation statement)

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“…Dideoxy sequencing, using synthetic primers, was done by the procedure of Hattori and Sakaki [16] with minor modifications. Plasmid DNA (1 pmol) was denatured in alkali and coprecipitated with primer (2-8 pmol) as described previously [17]. Subsequent operations were performed as described [15] except that 35S-dCTP (450 Ci/mmol) was used instead of [32P]dCTP, the C-reaction mix was changed to 83.2 pM each in dATP, dGTP, dTTP and ddCTP and the reaction temperature was 43 "C. A quarter of the individual reactions was analyzed on a 50-cm, 0.2-0.4-mm sequencing wedge gel (6 -8% acrylamide/8 M urea) run in Tris/borate [ll] at 68 'C with 2400 V applied.…”

Section: N a Sequencingmentioning

confidence: 99%

Molecular cloning and sequencing of a cDNA encoding the acyl carrier protein and its flanking domains in the mammalian fatty acid synthetase

Witkowski¹,

Naggert²,

Mikkelsen³

et al. 1987

Eur J Biochem

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Cloned cDNAs containing coding sequences for domains proximal to the carboxy terminus of the rat fatty acid synthetase have been isolated using an expression vector and domain-specific antibodies. The coding regions were assigned to specific domains of the multifunctional complex by identification of sequences coding for characterized peptide fragments and by recognition of sequences homologous to other monofunctional enzymes. Two clones contain the entire coding region for the acyl carrier protein domain. The sequence is flanked at the 3'-end by a region coding for the thioesterase domain and at the 5'-end by a sequence coding for a reductase, most likely the ketoreductase domain. Thus the ordering of these domain-coding regions in the fatty acid synthetase mRNA is established.The acyl carrier protein domain exhibits about 25% homology with that of the discrete monofunctional acyl carrier proteins of Escherichia coli, spinach and barley, the ketoreductase domain exhibits about 25% homology with bacterial dihydrofolate reductases and the active site of the thioesterase domain exhibits both primary and secondary structural features common to the serine proteases. These findings lend support to the hypothesis that the polyfunctional fatty acid synthetase probably arose by a complex evolutionary process involving fusion of genes coding for seven individual enzymes.In most simple bacteria and in plant chloroplasts the proteins which catalyze the de novo synthesis of fatty acids from malonyl-CoA exist as seven discrete monofunctional polypeptides [l]. However, in fungi the functional components are distributed between two non-identical polypeptides 121 and in animals they are integrated into a single multifunctional polypeptide 131. At present no convincing experimental evidence has been obtained which explains why or how different organizational forms of this enzyme system have evolved, although a number of theoretical arguments have been expounded [4, 51. As a first step to tackling this question we have initiated an analysis of the primary structure of the animal fatty acid synthetase. In this paper we report, for the first time, the cloning and sequencing of one of the component functional domains of a mammalian fatty acid synthetase, the acyl carrier protein. The sequence is compared with those of several acyl carrier proteins, which exist in lower life forms as separate monofunctional proteins, in order to assess the possibility that the proteins may have evolved from a common ancestral form. MATERIALS AND METHODS Bacterial strains and vectorsPhage Igt 11, Escherichia coli Y1090(r-) [dlacU169 proA+ dlon araD139 strA supF (trpC22: :TnlO) hsdR-hsdM' (pMC9)] and E. coli Y1089(r-) [dlacU169 proA' dlon ara D139 strA hflA (chr: :TnlO) hsdR-hsdM+ (pMC9)] were supplied by Promega Biotec (Madison, WI).

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Section: N a Sequencingmentioning

confidence: 99%