A cloned cDNA for duck malic enzyme detects abnormally large malic enzyme mRNAs in a strain of mice (Mod-1n) that does not express malic enzyme protein

Glynias, Manuel J.; Morris, Sidney M.; Fantozzi, Dominic A.; Winberry, L; Back, Donald W.; Goodridge, Alan G.

doi:10.1021/bi00310a011

Cited by 13 publications

(2 citation statements)

References 28 publications

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“…Recently, an mRNA species that hybridizes with a specific M od-l+ cDNA probe has been detected in M od-ln/ M od-ln tissue, suggesting that the m utant gene is transcribed. This mRNA is, however, larger than its wild-type counterpart (Glynias 1984;Sul 1984) and, intriguingly, can be translated into a correspondingly larger polypeptide in a rabbit reticulocyte system. Although the product of in vitro translation reacted with an antiserum against wild type enzyme (Sul et al 1984), it was not tested for retention of activity.…”

Section: Eployment Of Cells Within Tissuesmentioning

confidence: 92%

Clonal analysis of early mammalian development

Gardner

1985

Phil. Trans. R. Soc. Lond. B

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Various extrinsic markers have been used to label single cells in the early mouse embryo. However, they are appropriate only for short-term experiments because of their susceptibility to dilution. Studies on cell lineage and commitments have therefore depended mainly on exploiting genes as markers by combining cells from embryos that differ in genotype at particular loci. Tissue recombination and transplantation experiments using such indelible intrinsic markers have enabled the fate of different cell populations in the blastocyst to be determined with reasonable precision. The trophectoderm and inner cell mass (i.c.m.) give rise to distinct complementary groups of tissues in the later conceptus, as do the primitive endodermal and primitive ectodermal components of the more mature i.c.m. When cloned by blastocyst injection, single i.c.m. cells colonize only those parts of host conceptuses that are derived from their tissue of origin. Thus, while clonal descendants of early i.c.m. cells can contribute to all tissues other than those of trophectodermal origin, primitive endodermal and primitive ectodermal clones are restricted, respectively, to the extraembryonic endoderm versus all i.c.m. derivatives except the extraembryonic endoderm. Interestingly, individual primitive ectoderm cells can include both germ cells and somatic cells among their mitotic descendants. By using the genetically determined presence versus absence of cytoplasmic malic enzyme activity as a cell marker, the deployment of clones has been made visible in situ in whole-mount preparations of extraembryonic membranes. Very little mixing of donor and host cells was seen in either the endoderm of the visceral yolk sac or the mesodermal and ectodermal layers of the amnion. In contrast, mosaicism in the parietal endoderm was so fine grained that, in all except 1 of 15 fields from several specimens that were analysed, the arrangement of donor and host cells did not differ significantly from that expected on the basis of their random association.

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Section: Eployment Of Cells Within Tissuesmentioning

confidence: 92%

Clonal analysis of early mammalian development

Gardner

1985

Phil. Trans. R. Soc. Lond. B

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“…Subcloning of 'malic' enzyme cDNA into pUC-8 pDME-l ( Fig. la) contains the cDNA for duck liver 'malic' enzyme cloned into the PstI site of pBR322 (Glynias et al, 1984). It was digested with PstI and the products were separated by agarose-gel electrophoresis.…”

Section: Methodsmentioning

confidence: 99%

Duck liver ‘malic’ enzyme. Expression in Escherichia coli and characterization of the wild-type enzyme and site-directed mutants

Hsu

Glynias

Satterlee

et al. 1992

Biochemical Journal

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A cDNA for duck liver 'malic' enzyme (EC 1.1.1.40) was subcloned into pUC-8, and the active enzyme was expressed in Escherichia coli TG-2 cells as a fusion protein including a 15-residue N-terminal leader from beta-galactosidase coded by the lacZ' gene. C99S and R70Q mutants of the enzyme were generated by the M13 mismatch technique. The recombinant enzymes were purified to near homogeneity by a simple two-step procedure and characterized relative to the enzyme isolated from duck liver. The natural duck enzyme has a subunit molecular mass of approx. 65 kDa, and the following kinetic parameters for oxidative decarboxylation of L-malate at pH 7.0: Km NADP+ (4.6 microM); Km L-malate (73 microM); kcat (160 s-1); Ka (2.4 microM) and Ka' (270 microM), dissociation constants of Mn2+ at 'tight' (activating) and 'weak' metal sites; and substrate inhibition (51% of kcat. at 8 mM-L-malate). Properties of the E. coli-derived recombinant wild-type enzyme are indistinguishable from those of the natural duck enzyme. Kinetic parameters of the R70Q mutant are relatively unaltered, indicating that Arg-70 is not required for the reaction. The C99S mutant has unchanged Km for NADP+ and parameters for the 'weak' sites (i.e. inhibition by L-malate, Ka'); however, kcat. decreased 3-fold and Km for L-malate and Ka each increased 4-fold, resulting in a catalytic efficiency [kcat./(Km NADP+ x Km L-malate x Ka)] equal to 3.7% of the natural duck enzyme. These results suggest that the positioning of Cys-99 in the sequence is important for proper binding of L-malate and bivalent metal ions.

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Terminal differentiation in the avian uropygial gland. Accumulation of fatty acid synthase and malic enzyme in non-dividing cells

Jenik

Goodridge

1987

Cell Tissue Res.

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The secretory tissue of the uropygial gland is of the holocrine type, containing both dividing progenitor cells and lipid-filled differentiated cells. In this study, we examined the relationship between cell division and differentiation. The location of dividing cells was determined by autoradiography of tissue sections from ducklings injected intra-abdominally with 3H-thymidine. Only cells on the basal lamina of the tubules contained labeled nuclei. Dividing cells were distributed uniformly over the length of the tubules. Over the next five days, most of the labeled cells migrated to the lumen of the tubules and disappeared. Cells containing the "lipogenic" enzymes, fatty acid synthase and malic enzyme, were localized either immunocytochemically using affinity-purified antibodies or cytochemically using a specific assay for malic enzyme activity. Fatty acid synthase and malic enzyme were undetectable in dividing basal cells but present at high levels in differentiating and differentiated cells. Thus, basal cells lying along the basal lamina of the tubules were replacing lipid-laden cells that were continually sloughed into the lumens of the tubules. The signals for differentiation and enzyme accumulation appear to be linked to one another and to cessation of cell division.

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A cloned cDNA for duck malic enzyme detects abnormally large malic enzyme mRNAs in a strain of mice (Mod-1n) that does not express malic enzyme protein

Cited by 13 publications

References 28 publications

Clonal analysis of early mammalian development

Clonal analysis of early mammalian development

Duck liver ‘malic’ enzyme. Expression in Escherichia coli and characterization of the wild-type enzyme and site-directed mutants

Terminal differentiation in the avian uropygial gland. Accumulation of fatty acid synthase and malic enzyme in non-dividing cells

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