Demonstration, by renaturation in O'Farrell gels, of heterogeneity in Dictyostelium uridine diphosphoglucose pyrophosphorylase

Manrow, Richard E.; Dottin, Robert P.

doi:10.1016/0003-2697(82)90334-7

Cited by 24 publications

(9 citation statements)

References 14 publications

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“…This suggested that either low levels of Udpgp1 activity were still present, a second gene existed, or UDPGP activity was not required for development in Dictyostelium. Consistent with the second hypothesis, Manrow and Dottin (13) and Fishel et al (14) found that there appear to be two isoforms of UDPGP.…”

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confidence: 69%

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A Second UDP-glucose Pyrophosphorylase Is Required for Differentiation and Development in Dictyostelium discoideum

Bishop

Moon

Harrow

et al. 2002

Journal of Biological Chemistry

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Uridine diphosphoglucose pyrophosphorylase (UD-PGP) is a developmentally regulated enzyme in Dictyostelium discoideum, which is involved in trehalose, cellulose, and glycogen synthesis. Two independent UDPGP proteins are believed to be responsible for this activity. To determine the relative contributions of each protein, the genes encoding them were disrupted individually. Cells lacking the udpgp1 gene exhibit normal growth and development and make normal levels of cellulose. In agreement with these phenotypes, udpgp1 ؊ cells still have UDPGP activity, although at a reduced level. This supports the importance of the second UD-PGP gene. This newly identified gene, ugpB, encodes an active UDPGP as determined by complementation in Escherichia coli. When this gene is disrupted, cells undergo aberrant differentiation and development ending with small, gnarled fruiting bodies. These cells also have decreased spore viability and decreased levels of glycogen, whose production requires UDPGP activity. These phenotypes suggest that UgpB constitutes the major UDPGP activity produced during development. Sequence analysis of the two UDPGP genes shows that UgpB has higher homology to other eukaryotic UDPGPs than does UDPGP1. This includes the presence of 5 conserved lysine residues. Udpgp1 only has 1 of these lysines.

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confidence: 69%

“…In Dictyostelium, although the UDPGP transcripts are not temporally segregated, Manrow and Dottin (13) and Fishel et al (14) have reported the existence of UDPGP isozymes with distinct molecular weights and isoelectric points. These isozymes have differing patterns of activity in vegetative and stationary phase cells.…”

Section: Discussionmentioning

confidence: 99%

A Second UDP-glucose Pyrophosphorylase Is Required for Differentiation and Development in Dictyostelium discoideum

Bishop

Moon

Harrow

et al. 2002

Journal of Biological Chemistry

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“…The eluted antibodies were immediately neutralized by addition of 1M Tris HCl, pII 8.8, and concentrated in Centricon 100 microconcentrators (Amicon, Danvers, MA) for further analysis by IIF and immunoblotting. SDS-PAGE induces the denaturation of proteins (38,39), and several procedures for renaturing proteins before or after transfer to membranes have been proposed (40)(41)(42)(43)(44)(45)(46). We adapted some of these methods in trying to renature NuMA-2 antigen (see below) and to obtain immunoblot reactivity.…”

Section: Methodsmentioning

confidence: 99%

Two major autoantigen—Antibody systems of the mitotic spindle apparatus

Andrade

Chan

Peebles

et al. 1996

Arthritis & Rheumatism

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Objective. To characterize human autoantigenantibody systems related to the mitotic poles and spindles.Methods. Thirty-seven human sera with autoantibodies staining mitotic poles and spindles in indirect immunofluorescence (IIF) studies were further characterized by immunofluorescence on mitotic cells and by immunoblotting and immunoprecipitation. Clinical diagnoses meeting the American College of Rheumatology criteria were based on chart review and interview with the corresponding physicians.ResuUs. Two autoantibody systems reactive with mitotic poles and spindles were defined. Type 1 nuclear mitotic apparatus (NuMA-1) antibodies were identified in the serum of 30 patients. Interphase cells showed a fine, speckled, nuclear staining, while mitotic cells had bright staining of the rim of the centrosomes and light staining of the spindles proximal to the centrosomes. In telophase, the staining shifted from the centrosomes to the reforming nuclei. On immunoblotting, antiNuMA-1 sera reacted with a 210-kd protein. The reactivity of these sera was identified (with the aid of reference antibodies) as the previously described NuMA antigen-antibody system. Clinical information was available for only 17 of the 30 patients with antiNuMA-1; of these, 17 (53%) had clinical and lip biopsy findings that met the criteria for Sjiigren's syndrome.Publication 7668-MEM from The Scripps Research Institute.

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“…In two separate gels, the protein mixture was subjected to two-dimensional electrophoresis by the method of O'Farrell (19). The gels were stained separately, one with Coomassie blue and one for enzyme activity (22). Autoradiography showed that radioactive proteins comigrated with the purified proteins in both cases, thus confirming the identity of the immunoprecipitated polypeptides as CK-M (data not shown).…”

Section: Resultsmentioning

confidence: 58%

“…The radioactive translation products were compared to the pattern of CK from extracts of myogenic cell cultures. CK protein can be localized by activity after renaturation in two-dimensional gels: NaDodSO4/urea electrophoresis in the first dimension and isoelectric focusing in the second dimension (8,18,22). Myoblasts from 10-day chicken embryos were cultured for about 72 hr until just beyond the onset of fusion, at which time they were harvested for two-dimensional electrophoresis.…”

Section: Resultsmentioning

confidence: 99%

Molecular cloning of a DNA sequence complementary to creatine kinase M mRNA from chickens.

Schweinfest¹,

Kwiatkowski²,

Dottin³

1982

Proc. Natl. Acad. Sci. U.S.A.

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We have cloned and identified a DNA sequence complementary to the mRNA of creatine kinase (CK) isozyme M, although the mRNA is a minor species of the total mRNA in developing myoblasts. Poly(A)+RNA from breast and thigh muscle of 5-week-old chicks was enriched for CK mRNA by a novel procedure of sucrose gradient centrifugation in the presence of methylmercuric hydroxide. DNA complementary to this mRNA was inserted into pBR322, and colonies containing the recombinant plasmids were screened for the ability of the plasmid DNA to hybridize with and rescue CK mRNA from total muscle mRNA. Three plasmids, pCS195, pCS192, and pM35-4, could specifically rescue CK-M mRNA. CK-M mRNA was detected by in vitro translation and specific immunoprecipitation. The identity of the in vitro translation product was further confirmed by its migration in two-dimensional gels at the isoelectric point and molecular weight ofCK-M. The heterogeneity ofCK-M observed in vivo also was found upon translation of the CK-M mRNA which hybridizes to the plasmid.Differentiation of myoblasts into a functional myotube requires the synthesis not only of abundant contractile proteins but also of minor enzymatic proteins (1-3). Creatine kinase (CK), one such enzyme, is a dimer ofsubunit molecular weight 41,000 and is involved in the synthesis ofATP which is required for muscle contraction (4). One CK isozyme, the BB form, is present in many tissues and in undifferentiated myoblasts, but during myogenesis the synthesis of a second isozyme, the MM form, is induced (5-7). Recent reports have indicated that the MM and BB isozymes contain two subspecies which may be distinguished on high-resolution two-dimensional gels, but the genetic origin of this heterogeneity is unknown (8,9).In order to investigate the mechanisms that control the expression of these enzymes and the switching of isozyme forms, it is necessary first to isolate their genes. The isolation of genes that encode minor but important proteins requires refinements in the recombinant DNA protocols in order to enrich for the mRNAs and allow detection of these sequences.We report here the molecular cloning of DNA sequences complementary to CK-M mRNA. CK is identified by two criteria: its precipitation by specific antibodies against purified CK-M, and its mobility in two-dimensional gels. The heterogeneity that is observed in the subunits of CK-M in vivo is also found in the translation products of mRNA that hybridizes to the cloned plasmid DNA.MATERIALS AND METHODS RNA Isolation and Partial Purification of CK mRNA. Twenty-five grams of frozen thigh and breast muscle from 5-week-old chicks was ground to a powder under liquid nitrogen and was resuspended in 100 ml of 8 M guanidine-HCI (Heico)/ 10 mM Tris-HCl, pH 7.3, by stirring for 10 min at room temperature. An equal volume of H20-saturated phenol was added, the mixture was stirred for 10 min and centrifuged (25 min at 1,020 x g), and the supernatant was mixed with 4 vol of 100% ethanol. The suspension was chilled at -80°C for 1 hr to preci...

show abstract

Demonstration, by renaturation in O'Farrell gels, of heterogeneity in Dictyostelium uridine diphosphoglucose pyrophosphorylase

Cited by 24 publications

References 14 publications

A Second UDP-glucose Pyrophosphorylase Is Required for Differentiation and Development in Dictyostelium discoideum

A Second UDP-glucose Pyrophosphorylase Is Required for Differentiation and Development in Dictyostelium discoideum

Two major autoantigen—Antibody systems of the mitotic spindle apparatus

Molecular cloning of a DNA sequence complementary to creatine kinase M mRNA from chickens.

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