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ACCESSION NO: 0176827 SUBFILE: CRIS
PROJ NO: DEL00384 AGENCY: CSREES DEL
PROJ TYPE: HATCH PROJ STATUS: NEW
START: 01 OCT 1997 TERM: 30 SEP 2002 FY: 2000

INVESTIGATOR: Frick, H.; Keeler, C.; Burnside, J.

PERFORMING INSTITUTION:
PLANT & SOIL SCIENCES
UNIVERSITY OF DELAWARE
NEWARK, DELAWARE 19717

LACTOSE UTILIZATION IN THE ANGIOSPERM LEMNA MINOR L., AND ITS CALLUS

OBJECTIVES: 1.) Purify Lemna callus B-galactosidase to determine kinetics
and establish preferred substrate and obtain polyclonal antibody to this
purified enzyme protein. 2.) Clone CDNAs to include that for Lemna
B-galactosidase and recognize expression using the polyclonal of Objective
1.

APPROACH: 1.) Pooled callus and spent-agar extracts chromatographed
(Sepharose CL-4B, DEAE-Sepharose CL-6B/CM Sepharose CL-6B,
lactosyl-Sepharose 4-B), purity established by 2-D PAGE, and substrate
preference by standard kinetic analysis. 2.) Poly(A)+RNA via biotinylated
oligo (dt) probe; cDNA synthesis primed with oligo (dt) (Xhol) primer, ECOR
linkers then ligated, and cDNA ligated into lambda Zap II. Positive
phageclones recognized by indirect reaction to polyclonal antibody of
Objective 1. Excised plasmid DNA of positive clones to be sequenced and
used as Northern blot probes.

PROGRESS: 2000/01 TO 2000/12
Metabolism of exogenous carbohydrates by axenic, dark-grown Spirodela
punctata (monocot, duckmeat) meets several criteria for sugar sensing.
Unlike Arabidopis thaliana, Spirodela utilizes galactose to drive
heterotrophic growth and switches readily among sucrose, glucose, and
galactose. A Glycine max cDNA galactokinase clone has been verified and is
being used to probe poly-A RNA of Spirodela to obtain a homologous galK
cDNA clone. This will be used to establish whether catabolite repression by
glucose on galk mRNA transcription occurs in this angiosperm upon
carbohydrate shifting. M1 seed of Arabidopsis (EMS) has been selected for
germination in 75 mM D-mannose plus heterotrophic growth in darkness on 2
percent glucose plus greening on glucose in the light in hopes of
recognizing a gain-of-function mutant for mannose-6-phosphate activity --
mitigating against the possibility that hexokinase is at the start of the
sugar sensing transduction sequence.

PUBLICATIONS: 2000/01 TO 2000/12
No publications reported this period
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