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Query: EC:3.2.1.23 (beta-galactosidase)
 14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A cDNA expression strategy was used to localize amino acid sequences which were specific for fast, as opposed to slow, isoforms of the chicken skeletal muscle myosin heavy chain (MHC) and which were conserved in vertebrate evolution. Five monoclonal antibodies (mAbs), termed F18, F27, F30, F47, and F59, were prepared that reacted with all of the known chicken fast MHC isoforms but did not react with any of the known chicken slow nor with smooth muscle MHC isoforms. The epitopes recognized by mAbs F18, F30, F47, and F59 were on the globular head fragment of the MHC, whereas the epitope recognized by mAb F27 was on the helical tail or rod fragment. Reactivity of all five mAbs also was confined to fast MHCs in the rat, with the exception of mAb F59, which also reacted with the beta-cardiac MHC, the single slow MHC isoform common to both the rat heart and skeletal muscle. None of the five epitopes was expressed on amphioxus, nematode, or Dictyostelium MHC. The F27 and F59 epitopes were found on shark, electric ray, goldfish, newt, frog, turtle, chicken, quail, rabbit, and rat MHCs. The epitopes recognized by these mAbs were conserved, therefore, to varying degrees through vertebrate evolution and differed in sequence from homologous regions of a number of invertebrate MHCs and myosin-like proteins. The sequence of those epitopes on the head were mapped using a two-part cDNA expression strategy. First, Bal31 exonuclease digestion was used to rapidly generate fragments of a chicken embryonic fast MHC cDNA that were progressively deleted from the 3' end. These cDNA fragments were expressed as beta-galactosidase/MHC fusion proteins using the pUR290 vector; the fusion proteins were tested by immunoblotting for reactivity with the mAbs; and the approximate locations of the epitopes were determined from the sizes of the cDNA fragments that encoded a particular epitope. The epitopes were then precisely mapped by expression of overlapping cDNA fragments of known sequence that covered the approximate location of the epitopes. With this method, the epitope recognized by mAb F59 was mapped to amino acids 211-231 of the chicken embryonic fast MHC and the three distinct epitopes recognized by mAbs F18, F30, and F47 were mapped to amino acids approximately 65-92. Each of these epitope sequences is at or near the ATPase active site.
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PMID:Evolutionarily conserved sequences of striated muscle myosin heavy chain isoforms. Epitope mapping by cDNA expression. 247 86

We show that a fusion gene, containing the promoter and 5'-noncoding region of a Dictyostelium discoideum actin 6 gene linked to the Escherichia coli beta-galactosidase (beta Gal) gene (lacZ), directs the production of functionally active beta Gal in D. discoideum and that the enzyme can be detected by staining in situ; a procedure which will be of great value in analyzing cell-type-specific gene expression. We illustrate this by fusing lacZ to the promoter of the prespore-specific gene, D19, and localizing expressing cells in migrating slugs. Optimal expression requires the inclusion of termination and polyadenylylation signals and we describe pDDlac, a vector containing a multiple cloning site upstream from a lacZ-Dictyostelium terminator fusion, which can be used to analyze regulated promoters.
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PMID:Optimization and in situ detection of Escherichia coli beta-galactosidase gene expression in Dictyostelium discoideum. 251 30

We screened a cDNA library of the primitive eukaryote Dictyostelium discoideum constructed in the expression vector lambda gt11 with a specific antiserum directed against S-adenosyl-L-homocysteine hydrolase (AdoHcy hydrolase) and isolated cDNA clones coding for fusion proteins with beta-galactosidase. The identity of the largest of these clones was further assessed by analysis of hybrid-selected in vitro translation products. Using the cDNA as a probe, we showed that only one gene coding for AdoHcy hydrolase is present per genome and that a single transcript of 1.6 kb could be detected at various stages of differentiation. The nucleotide sequence of the cDNA was determined. It corresponds to the carboxyterminal half of the protein whose primary structure is highly homologous to the AdoHcy hydrolase from rat liver. The significance of this strong conservation of AdoHcy hydrolase in the course of evolution is discussed.
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PMID:Cloning of a cDNA for the S-adenosyl-L-homocysteine hydrolase from Dictyostelium discoideum. 313

Using antibodies specific for the 3',5'-cyclic AMP (cAMP) cell surface receptor of Dictyostelium discoideum, we have screened lambda gtll expression libraries and isolated a series of cDNAs derived from cAMP receptor mRNA during early development. The identity of the cDNA clones was verified by multiple criteria: 1) beta-galactosidase fusion proteins synthesized by isolated cDNA clones stain intensely with cAMP receptor directed antiserum, 2) these fusion proteins affinity purify antibodies specific for the cAMP receptor, 3) the cDNA probes hybridize to a 2 kb mRNA whose change in relative level of abundance during development parallels that of receptor mRNA as assayed by in vitro translation, 4) the 2 kb mRNA size equals that of receptor mRNA as determined by in vitro translation of size fractionated poly (A)+ RNA, and 5) RNA transcribed in vitro from cDNAs containing the entire protein-coding region produces a polypeptide by in vitro translation with an apparent molecular weight in close agreement with that of nascent cAMP receptor protein produced by in vitro translation of cellular RNA. The DNA sequence predicts an open reading frame of 392 amino acids. The deduced amino acid sequence contains seven domains enriched in hydrophobic residues. A model is proposed in which the cAMP cell-surface receptor traverses the lipid bilayer seven times in a pattern similar to that of other receptors, such as rhodopsin, which interact with G-proteins. The structural similarities suggest a gene family of related surface receptors from such evolutionarily diverse species as Dictyostelium, yeast, and mammals.
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PMID:Structure and expression of the cAMP cell-surface receptor. 324 22

EB4 is one of several cloned cDNAs that is expressed as mRNA only after the aggregation stage of Dictyostelium discoideum differentiation and exclusively in prespore and spore cells (E. Barklis and H. F. Lodish, Cell 32:1139-1148, 1983). We have isolated the unique genome fragment corresponding to the 5' portion of the EB4 message and the EB4 promoter. The EB4 transcript has an unusually long, G + C-rich, 5' noncoding region, but initiates at several start sites within a region of DNA that is 96% A + T. The sequence GTGGTGG, along with slight variations, occurs several times in the promoter. We have used the EB4 promoter to drive the transcription of an EB4/beta-galactosidase fusion transcript in yeast cells. Although the cap sites of the fused transcript in yeast cells are located in the region where multiple EB4 transcripts are initiated in Dictyostelium, the unregulated expression of the fusion transcript in yeast does not mimic the normal regulated pattern of EB4 mRNA expression in D. discoideum.
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PMID:Structure of the promoter of the Dictyostelium discoideum prespore EB4 gene. 389 36

Monoclonal antibodies were raised against two soluble, galactose-binding lectins from cells of Dictyostelium discoideum, discoidin I and II. These antibodies reacted not only with both discoidins, but also with a plasma membrane glycoprotein of aggregation competent cells, called contact site A, and with two carbohydrate-binding proteins of E. coli, beta-galactosidase and lac repressor. The possibility that the antibody recognizes a structure common to different carbohydrate-binding proteins is discussed. The two carbohydrate-binding proteins of E. coli share with discoidin I the sequence -Ser-X-X-Ile-His(Pro)-Pro(His)-Leu-Thr- which might be responsible for the cross-reactivity.
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PMID:Monoclonal antibody against cytoplasmic lectins of Dictyostelium discoideum: cross-reactivity with a membrane glycoprotein, contact site A, and with E. coli beta-galactosidase and lac repressor. 620 16

The ability of 24 systematically modified analogues of adenosine 3',5'-monophosphate (cAMP) to enhance the synthesis of beta-galactosidase in glucose-repressed Escherichia coli strains KNBL 1001 and cpd- Crookes has been investigated. The properties of the analogues in comparison with cAMP are, with only two exceptions, alike in both strains. Two analogues, 7-deazaadenosine 3',5'-monophosphate (i.e. tubercidin 3',5'-monophosphate) and (Rp)-adenosine 3',5'-monothionophosphate, exhibit higher biological activity than cAMP. The latter analogue is 50-fold more active in both strains. Three analogues showed activities comparable to cAMP, four analogues were less active and 12 analogues were unable to antagonize catabolite repression. Structure-activity correlations showed that the 2'OH-, 3'O-, 5'O-, the negative charge and the 6-amino group cannot be modified without losing biological activity in vivo, while the N-1 and N-7 in adenine are not essential. The interaction with the catabolite gene activator protein is stereoselective for an unmodified axial exocyclic oxygen. The results are compared to those obtained with cAMP analogues in E. coli in vitro and those obtained with the same analogues in protein-kinase systems and Dictyostelium species. The model of McKay et al. [McKay, D.B., Weber, J.T. and Steitz, T.A. (1982) J. Biol. Chem. 257, 9518-9524] proposed for distinct chemical interactions of cAMP with the catabolite gene activator protein is discussed and supplemented by additional hydrogen bond interactions.
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PMID:Investigations on stimulation of lac transcription in vivo in Escherichia coli by cAMP analogues. Biological activities and structure-activity correlations. 631 29

Plasma membrane glycoconjugates, enzymatically labelled with [3H]galactose, were used as an autoradiographic membrane marker for a morphometric analysis of membrane traffic during fluid phase pinocytosis in the amoeba, Dictyostelium discoideum. The fraction of grains associated with the plasma membrane decreased exponentially from 99% for cells fixed directly after labelling on the cell surface, to a steady-state value of 45% after about 1 h of pinocytotic activity. The complementary fraction of grains was observed on vacuolar membranes. Only after a lag of about 20 min, a small but significant fraction (3%) of the total grains, was found in the region of the Golgi membranes. During two subsequent doublings of cell number, over a period of 24 h, label was lost into the medium at a constant rate of 1% per h. The cell bound label remained fully membrane bound over the entire period. The beta(1-4) linkage was not noticeably modified, as judged by its susceptibility to hydrolytic release by beta-galactosidase. An analysis by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) showed an identical labelling pattern for total membrane fractions when prepared directly after labelling or after 24 h of membrane flow.
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PMID:Membrane traffic in Dictyostelium discoideum: plasma membrane glycoconjugates internalized and recycled during fluid phase pinocytosis enter the Golgi complex. 664 36

Several lysosomal glycosidase activities were examined in vitro during heat-induced germination of Dictyostelium discoideum spores and were found not to be coordinately controlled. The level of beta-glucosidase activity increased significantly during the emergence stage of germination. Both alpha-glucosidase and N-acetyl-beta-glucosaminidase activities remained relatively constant until postemergence, when they increased slightly; alpha-mannosidase activity decreased during all stages of germination. The activity of beta-galactosidase increased slightly during spore swelling, fell below the level initially found in spores at zero time, and increased slightly during postemergence. The expression of all of these enzyme activities, except the increase in beta-galactosidase, appeared to require protein synthesis. Spores in the lag phase of germination which were exposed to severe environmental stress were deactivated and exhibited reduced levels of alpha-glucosidase, beta-glucosidase, and N-acetyl-beta-glucosaminidase activities. Prolonged heat activation treatment reduced the levels of lysosomal glycosidase activities in postactivated spores but did not change the subsequent enzyme patterns during the spore-swelling and emergence stages of germination.
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PMID:Expression of glycosidase activities during germination of Dictyostelium discoideum spores. 676 80

We have been able to hybridize nonradioactive probes from cell-type-specific genes to fixed whole-mounts prepared at the mound, slug, and culminant stages of Dictyostelium development. The cellular patterns of labeling with probes from the prespore gene, cotB, and the prestalk genes, ecmA and ecmB, confirmed the patterns seen in strains carrying reporter constructs in which the regulatory regions of these genes drive beta-galactosidase. This technique permits the direct observation of protein synthetic capacity from characterized genes without the need of generating transformed lines carrying specific reporter constructs. Moreover, the pattern is not complicated by a previous developmental history of gene expression.
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PMID:Whole-mount in situ hybridization of cell-type-specific mRNAs in Dictyostelium. 755 3


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