Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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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)

Inaccurate protein synthesis produces unstable beta-galactosidase, whose activity is rapidly lost at high temperature. Erythromycin, lincomycin, clindamycin, and celesticetin were shown to counteract the error-inducing effects of streptomycin on beta-galactosidase synthesized in the antibiotic-hypersensitive Escherichia coli strain DB-11 Met-. Newly synthesized beta-galactosidase was more easily inactivated by high temperatures when synthesized by bacteria partially starved for arginine, threonine, or methionine. Simultaneous treatment with erythromycin or lincomycin yielded beta-galactosidase that was inactivated by high temperatures less easily than during starvation alone, an effect attributed to stimulation of ribosome editing. When synthesized in the presence of canavanine, beta-galactosidase was inactivated by high temperature more easily but this effect could not be reversed by erythromycin. The first arginine in beta-galactosidase occurs at residue 13, so the effect of erythromycin during arginine starvation is probably to stimulate dissociation of erroneous peptidyl-tRNAs of at least that length. Correction of errors induced by methionine starvation is probably due to stimulation of dissociation of erroneous peptidyl-tRNAs bearing peptides at least 92 residues in length. All the effects of erythromycin or the tested lincosamides on protein synthesis are probably the result of stimulating the dissociation from ribosomes of peptidyl-tRNAs that are erroneous or short.
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PMID:Erythromycin, lincosamides, peptidyl-tRNA dissociation, and ribosome editing. 817 19

We have isolated two yeast genes, KIN1 and KIN2, by their homology to the protein kinase family of viral oncogenes. Previous studies have identified the yeast KIN1 gene product (pp145KIN1) as a 145 kilodalton (kDa) phosphoprotein with serine/threonine-specific protein kinase activity. To identify and biochemically characterize the KIN2 gene product, antibodies were raised against a bacterial beta-galactosidase/KIN2 fusion polypeptide. In vivo, the KIN2 gene product is a 145 kDa phosphoprotein, pp145KIN2. In immune complexes, pp145KIN2 demonstrates serine/threonine protein kinase activity, transferring phosphate from [gamma-32P]ATP to either itself or the exogenously added substrates alpha-casein, acid-denatured enolase, or phosvitin. In vitro, kinase activity is dependent on either Mn2+ or Mg2+ ions. Both enzymes, pp145KIN1 and pp145KIN2, prefer ATP over GTP as their phosphoryl donor. Since a new class of yeast protein kinases has been identified which are serine/tyrosine-specific, we analysed a wide range of substrates to see if any could be phosphorylated by pp145KIN1 or pp145KIN2 on tyrosine residues. Both enzymes phosphorylate alpha-casein, acid-denatured enolase, and phosvitin on serine and threonine residues. Neither enzyme could phosphorylate tyrosine residues even though good substrates for tyrosine-specific kinases such as enolase, angiotensin II, and the synthetic polymer GLU80TYR20 were used. The biochemical analysis of KIN2 kinase activity shows remarkable similarity to that of its most closely related yeast kinase, KIN1. It remains to be seen if these two yeast protein kinases share any functional relationships or substrates in vivo.
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PMID:Characterization of the KIN2 gene product in Saccharomyces cerevisiae and comparison between the kinase activities of p145KIN1 and p145KIN2. 820 45

Egr-1 is an immediate-early response gene induced transiently and ubiquitously by mitogenic stimuli and also regulated in response to signals that initiate differentiation. The Egr-1 gene product, a nuclear phosphoprotein with three zinc fingers of the Cys2His2 class, binds to the sequence CGCCCCCGC and transactivates a synthetic promoter construct 10-fold in transient-transfection assays. We have analyzed the structure and function of the Egr-1 protein in detail, delineating independent and modular activation, repression, DNA-binding, and nuclear localization activities. Deletion analysis, as well as fusions to the DNA-binding domain of GAL4, indicated that the activation potential of Egr-1 is distributed over an extensive serine/threonine-rich N-terminal domain. In addition, a novel negative regulatory function has been precisely mapped 5' of the zinc fingers: amino acids 281 to 314 are sufficient to confer the ability to repress transcription on a heterologous DNA-binding domain. Specific DNA-binding activity was shown to reside in the three zinc fingers of Egr-1, as predicted by homology to other known DNA-binding proteins. Finally, nuclear localization of Egr-1 is specified by signals in the DNA-binding domain and basic flanking sequences, as determined by subcellular fractionation and indirect immunofluorescence. Basic residues 315 to 330 confer partial nuclear localization on the bacterial protein beta-galactosidase. A bipartite signal consisting of this basic region in conjunction with either the second or third zinc finger, but not the first, suffices to target beta-galactosidase exclusively to the nucleus. Our work shows that Egr-1 is a functionally complex protein and suggests that it may play different roles in the diverse settings in which it is induced.
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PMID:A novel repression module, an extensive activation domain, and a bipartite nuclear localization signal defined in the immediate-early transcription factor Egr-1. 833 1

The involvement of serine/threonine protein phosphatases in signaling pathways which modulate the activity of the transcription factor AP-1 was examined. Purified protein phosphatase types 1 (PP1) and 2A (PP2A) were microinjected into cell lines containing stably transfected lacZ marker genes under the control of an enhancer recognized by AP-1. Microinjection of PP2A potentiated serum-stimulated beta-galactosidase expression from the AP-1-regulated promoter. Similarly, transient expression of the PP2A catalytic subunit with c-Jun resulted in a synergistic transactivation of an AP-1-regulated reporter gene. PP2A, but not PP1, potentiated serum-induced c-Jun expression, which has been previously shown to be autoregulated by AP-1 itself. Consistent with these results, PP2A dephosphorylated c-Jun on negative regulatory sites in vitro, suggesting one possible direct mechanism for the effects of PP2A on AP-1 activity. Microinjection of PP2A had no effect on cyclic AMP (cAMP)-induced expression of a reporter gene containing a cAMP-regulated promoter, while PP1 injection abolished cAMP-induced gene expression. Taken together, these results suggest a specific role for PP2A in signal transduction pathways that regulate AP-1 activity and c-Jun expression.
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PMID:Protein phosphatase 2A potentiates activity of promoters containing AP-1-binding elements. 838 5

Raf-1 is a serine/threonine specific kinase that integrates signaling by a large number of mitogens to elicit a transcriptional response in the nucleus. Activated Raf-1 phosphorylates and activates MAPK/ERK kinase Mek), thus initiating the Mek--> MAP kinase cascade, which ultimately results in the phosphorylation and activation of transcription factors by MAP kinase. Here we have characterized the mechanism by which monoclonal antibody URP26K, which binds to an epitope in the Raf-1 kinase domain, inhibits intracellular signal transduction. This antibody preferentially immunoprecipitated the underphosphorylated, non-activated form of Raf-1 from quiescent cells. Baculovirus-expressed Raf-1 immunoprecipitated with URP26K was largely refractory to phosphorylation and activation mediated by protein kinase C (PKC)alpha or the tyrosine kinase Lck. In addition, URP26K reduced the binding of Raf-1 to its substrate Mek in vitro, but did not disturb the association of Raf-1 with Ras. Microinjection of URP26K into Rat-1 cells blocked DNA synthesis initiated by serum, insulin and various purified growth factors, but it did not block DNA synthesis initiated by v-ras. Microinjected URP26K also impaired the expression of stably transfected beta-galactosidase reporter genes regulated by minimal promoter elements. These results demonstrate, (i) that the URP26K monoclonal antibody inhibits Raf-1 by preventing activating Raf-1 phosphorylation and/or association with its substrate Mek, (ii) that inhibition of Raf-1 by URP26K does not interfere with Ras-induced DNA synthesis. In contrast to dominant negative Raf-1 mutants, which also block Ras signaling by binding to the Ras effector domain, antibody mediated Raf-1 inhibition thus reveals a branchpoint of mitogenic signaling at the level of Ras.
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PMID:Inhibition of Raf-1 signaling by a monoclonal antibody, which interferes with Raf-1 activation and with Mek substrate binding. 880 5

Metabolic labelling of Plasmodium falciparum parasites with [3H]GlcN, [3H]Man, [3H]Gal and [3H]ethanolamine, and subsequent purification by SDS-PAGE of the labelled material provided effective labelling of the MSP-1, 195 kDa, and MSP-2, 42-53 kDa, glycoproteins. Reductive beta-elimination of the MSP-2 released from the gel consisted of glycopeptides containing labelled sugars. Processing of the eliminated components and identification of the sugar residues demonstrated the presence of N-acetylglucosaminitol and N-acetylgalactosaminitol amongst other labelled sugars. Reductive beta-elimination with sodium hydroxide-sodium borotritide-borohydride showed the presence of glucosaminitol and alanine in the hydrolysis products. The MSP-2 was retained on solid phase wheat-germ agglutinin and was released from the lectin by treatment with GlcNAc. Upon treatment with O-glycanase the MSP-2 glycoprotein released labelled amino sugar, and derived oligosaccharides on treatment with exoglycosidases released labelled components corresponding to the metabolically incorporated sugars. Labelled Gal was incorporated into the MSP-2 glycoprotein using [3H]UDP-Gal and galactosyltransferase. The galactosylated glycoprotein released labelled Gal upon treatment with beta-galactosidase. The results of the present study suggest that the carbohydrate chains of the MSP-2 glycoprotein are attached to the protein backbone via GlcNAc- and GalNAc-serine/threonine in O-glycosyl linkage and the glycoprotein has terminal GlcNAc and Gal residues. The carbohydrate moieties of MSP-2, glycoprotein consist mainly of short chains linked to the protein core.
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PMID:Carbohydrate moiety of Plasmodium falciparum glycoproteins: the nature of the carbohydrate-peptide linkage in the MSP-2 glycoprotein. 935 84

A 3135 bp DNA segment downstream of the spl gene on the Bacillus subtilis chromosome was cloned and its nucleotide sequence determined. An open reading frame capable of encoding a putative protein of 654 amino acids with a calculated molecular mass of 72.1 kDa was identified. The deduced amino acid sequence was similar to the McpA and McpB proteins of B. subtilis. McpA and McpB encode different methyl-accepting chemotaxis proteins (MCPs). A mutant strain containing an antibiotic resistance DNA cassette inserted into the region containing the MCP-like reading frame suffered a complete loss of taxis to the amino acids cysteine, proline, threonine, glycine, serine, lysine, valine and arginine. The open reading frame was designated mcpC. The wild-type and an mcpC mutant strain were analysed for their content of methylated proteins and it was found that mcpC encodes a methylated membrane protein that has previously been designated H3. These results show that mcpC encodes a third MCP in B. subtilis. The transcription start site upstream of the mcpC gene was determined by primer extension analysis and it was found to be preceded by a potential promoter sequence that is recognized by the sigma D form of RNA polymerase. The level of beta-galactosidase expressed from a transcriptional mcpC-lacZ fusion was increased threefold when cells entered the stationary phase. No beta-galactosidase could be detected in a sigD genetic background.
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PMID:Functional and genetic characterization of mcpC, which encodes a third methyl-accepting chemotaxis protein in Bacillus subtilis. 935 24

In the filamentous fungus Neurospora crassa during conditions of sulfur limitation, CYS3, a major positive-acting regulatory protein, turns on the expression of an entire set of genes which encode permeases and enzymes involved in the acquisition of sulfur from environmental sources. CYS3 functions as a homodimeric protein and possesses a b-Zip domain that confers sequence-specific DNA binding. Expression of various hybrid GAL4-CYS3 fusion proteins in yeast was used to detect regions involved in gene activation. An amino-terminal serine/threonine-rich domain of CYS3 alone strongly activated expression of beta-galactosidase, the yeast reporter. Moreover, mutant CYS3 proteins with amino-acid substitutions in this region that showed increased expression in Neurospora also displayed an enhanced activation potential in yeast. The cys-3 gene of the exotic N. crassa Mauriceville strain and of N. intermedia were cloned and demonstrated to be functional for gene activation and for sulfur-mediated regulation by complementation of a loss-of-function cys-3 mutation. The amino-terminal serine/threonine-rich region is highly conserved in these two CYS3 proteins, in agreement with the possibility that it serves as the activation domain. Surprisingly, an extended promoter region of the cys-3 gene in the Mauriceville strain and in N. intermedia was very well conserved with that of the standard N. crassa gene, including the presence of three CYS3-binding sites possibly involved in autogenous control. Results are presented which indicate that synthesis of the CYS3 regulatory protein is highly regulated and can be detected in the nucleus of cells subjected to sulfur de-repression, but is not found in the nucleus or the cytoplasm of S-repressed cells. The amino-acid substitutions of the CYS3 protein present in a temperature-sensitive cys-3 mutant and in a second-site revertant of a cys-3 null mutation are presented and are shown to affect their DNA-binding activities.
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PMID:Functional analysis of different regions of the positive-acting CYS3 regulatory protein of Neurospora crassa. 964 2

A recessive mutation, aarG1, has been identified that resulted in an 18-fold increase in the expression of beta-galactosidase from an aac(2')-lacZ fusion. Transcriptional fusions and Northern blot analysis demonstrated that the aarG1 allele also resulted in a large increase in the expression of aarP, a gene encoding a transcriptional activator of aac(2')-Ia. The effects of aarG1 on aac(2')-Ia expression were mediated by aarP-dependent and -independent mechanisms. The aarG1 allele also resulted in a multiple antibiotic resistance (Mar) phenotype, which included increased chloramphenicol, tetracycline and fluoroquinolone resistance. This Mar phenotype also resulted from aarP-dependent and -independent mechanisms. Sequence analysis of the aarG locus revealed the presence of two open reading frames, designated aarR and aarG, organized in tandem. The putative AarR protein displayed 75% amino acid identity to the response regulator PhoP, and the AarG protein displayed 57% amino acid identity to the sensor kinase PhoQ. The aarG1 mutation, a C to T substitution, resulted in a threonine to isoleucine substitution at position 279 (T279I) in the putative sensor kinase. The AarG product was functionally similar to PhoQ, as it was able to restore wild-type levels of maganin resistance to a Salmonella typhimurium phoQ mutant. However, expression of the aarP and aac(2')-Ia genes was not significantly affected by the levels of Mg2+ or Ca2+, suggesting that aarG senses a signal other than divalent cations.
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PMID:A regulatory cascade involving AarG, a putative sensor kinase, controls the expression of the 2'-N-acetyltransferase and an intrinsic multiple antibiotic resistance (Mar) response in Providencia stuartii. 968 Feb 22

The activity of the medaka beta-actin promoter as a ubiquitous expression vector in transgenic medaka was examined using complementary DNA of the green fluorescent protein (GFP). Plasmid pOBA-GFP contained both the medaka beta-actin promoter and cDNA of the wild-type GFP, while pOBA-hGFP contained the medaka beta-actin promoter and cDNA of the mutant GFP in which serine was substituted for threonine at position 65 and codon usage was humanized to promote translation in vertebrate cells. The ApaI-SmaI fragment of both plasmids was microinjected into the nuclei of oocytes or the cytoplasm of embryos at the one-cell stage. The gene expression was detected, using a fluorescent stereomicroscope, from early stages of development to 1 week after hatching. The expression of the wild-type GFP was detected in early embryos, in the yolk sac and in small portions of the muscle and epidermis. This expression pattern was similar to that of the Escherichia coli beta-galactosidase reporter gene (lacZ), driven by the medaka beta-actin promoter, which was examined in our previous studies. The mutant GFP was expressed in early embryos and in many tissues such as the epidermis, blood vessels, muscle, notochord, fin ray, gut, eyes, and yolk sac, and the fluorescence was much stronger than that of the wild-type GFP. Thus, the usefulness of the medaka beta-actin promoter as a ubiquitous expression vector was confirmed using the mutant GFP as a reporter gene.
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PMID:Usefulness of the medaka beta-actin promoter investigated using a mutant GFP reporter gene in transgenic medaka (Oryzias latipes). 970 11


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