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| James Y. Wee (Masters Candidate in Biochemistry/Molecular Biology.) |
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James' research deals with the mode of assimilation of naphthalene by P. fluorescens. His ultimate objective is to identify a membrane associated protein that is specifically involved in naphthalene assimilation. However, his approach has been different from that taken by Tracy. Since specific transport proteins for naphthalene would be expected to be localized in the inner and/or outer membrane of a Gram negative organism, James initiated his studies by developing a procedure to resolve the inner and outer membranes from P. fluorescens and then compared the membrane protein profiles in the purified inner and outer membranes of glucose and naphthalene grown cells by SDS-PAGE. |
| The purified membranes were obtained by French pressure cell disruption of the glucose and naphthalene grown cells followed by the preparation of a crude membrane fraction by centrifugation at 106,000 x g for an hour. This crude membrane fraction was then resolved into inner and outer membranes by centrifugation through a linear sucrose gradient. |
P.fluorescens strain Uper-1 exposed to naphthalene after 48 hours. |
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membrane pellet after 1 hour of ultr-centrifugation at 106,000xg. Two tubes on the contain membrane pellet and the right tube contains the supernatant. |
Two membrane bands appeared after an 18-hour centrifugation at 36,000 rpm in a SW-41 rotor. The upper band has been putatively identified as cytoplasmic membrane and the lower membrane band as outer membrane. |
| The appearance of the naphthalene grown cells in naphthalene medium, as well as the appearance of the membranes from these cells following sucrose density gradient centrifugation, are shown at the left. SDS-PAGE analysis of these membrane preparations revealed a variety of differences in the protein compositions of both the outer and inner membranes from glucose and naphthalene grown cells. In particular, the outer membranes of naphthalene grown cells were shown to posses a 33kd protein that was not present in the same cells grown on glucose. Since most of the proteins in the outer membrane of a Gram negative organism have a transport or recognition function, James selected this 33kd protein for further studies. This protein was transferred from the SDS-PAGE to a PVDF membrane and this membrane was then used for commercial N-terminal sequencing. These studies yielded the first 30 amino acids from the N-terminus and this sequence was found to be identical with the N-terminal sequence from the outer membrane protein OprF. |
| This protein (OprF) is known to exist in pseudomonads, but it's function(s) haven't been completely defined. However, an oligonucleotide primer was developed using the N-terminal amino acid sequence and James has recently used this primer to produce PCR products from genomic DNA as template. These products have been cloned into pGEM-T and several transformants have been selected for further evaluation. The ultimate objective is to obtain the complete gene sequence so that the relationship of this protein to naphthalene assimilation can be studied. |
SDS-PAGE analysis of purified membrane fractions from glucose-adapted (G) and naphthalene- adapted (N) cells. The extreme right lane are the MW markers. From the left: GI, NI, LB-adapted membrane I, G II, N II, LB II, N I, and N II |
