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)

We have developed an ELISA which detects, with high specificity, antibodies against a major surface protein of P. falciparum merozoites which is a processing product of the precursor glycoprotein gp190. This assay can be used in the diagnosis of acute malaria in individuals with primary infection. Two partial sequences of gp190 were expressed in E. coli as beta-galactosidase (beta-Gal) fusion proteins. The same sequences fused to chloramphenicol acetyltransferase (CAT) or mouse dihydrofolate reductase (DHFR) react with high frequency when sera of acute malaria patients are analyzed in immunoblots. Antibodies from such sera crosslink, via their antigen binding sites, the beta-Gal fusions to the corresponding CAT or DHFR fusions adsorbed to a solid phase as demonstrated by the captured beta-Gal activity. The assay is highly specific, shows extremely low cut off values and should therefore be widely applicable.
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PMID:A new tool for the serodiagnosis of acute Plasmodium falciparum malaria in individuals with primary infection. 266 17

Hybrid genes were constructed. One, ompA153-dfr, encoded the precursor of the 325 residue Escherichia coli outer membrane protein OmpA up to residue 153 which was fused to the complete 186-residue dihydrofolate reductase of the mouse. The other, ompA219-lacZ, coded for the same precursor up to residue 219 which was fused to 1017 COOH-terminal residues of the 1023-residue subunit of the beta-galactosidase of E. coli. Full expression of the ompA153-dfr gene caused accumulation of its precursor and of that of the chromosomally encoded OmpA protein. When the amount of product was reduced, no pro-OmpA and very little pro-hybrid protein accumulated. The precursor was processed and the mature protein was fully accessible to trypsin in permeabilized cells. Expression of the ompA219-lacZ gene led to the presence of the hybrid protein at only 20-30% of the amount expected. About 20% of it appeared to be incorporated in the outer membrane. All of the hybrid was quantitatively accessible to trypsin in permeabilized cells. When the hybrid gene was overexpressed, the protein was found associated with the plasma membrane in the cytosol. It is concluded that both beta-galactosidase and dihydrofolate reductase could quantitatively traverse the plasma membrane, provided the amounts synthesized were sufficiently small.
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PMID:Dihydrofolate reductase (mouse) and beta-galactosidase (Escherichia coli) can be translocated across the plasma membrane of E. coli. 314 14

The transfer of the adenovirus 5 E1A gene into brain cells from rat embryos led to the establishment of phenotypically untransformed clonal glial cell lines. Some lines displayed properties of oligodendrocyte-astrocyte progenitors. Their differentiation involved several steps that were dependent on culture conditions and cell interactions. Subsequently, a few lines were cotransfected with a vector carrying a modified dihydrofolate reductase gene and with the Escherichia coli lacZ gene. After selection for resistance to methotrexate, cell lines were derived that stably expressed the lacZ gene. These cells were individually detectable by histochemical staining for beta-galactosidase activity, even in the presence of other cells. These results suggest that this type of cell line could be of interest for further in vitro, and possibly transplant, studies of the differentiation and interactions of glial cells.
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PMID:Immortalization of bipotential glial progenitors and generation of permanent "blue" cell lines. 314 80

A shuttle vector has been constructed by fusing the Bacillus subtilis trimethoprim-resistance-carrying (TpR) plasmid pNC601 with the Escherichia coli plasmid pBR322. The resultant plasmid pNBL1 can replicate in both B. subtilis and E. coli, conferring Tp resistance on both cells and ampicillin resistance (ApR) on E. coli. The B. subtilis dihydrofolate reductase operon (dfr) on pNC601 and therefore on pNBL1 consists of the thymidylate synthase B gene (thyB) and the TpR-dihydrofolate reductase gene lacking the C-terminal seven codons (designated as drfA' as compared with the complete dfrA gene). A direct-expression vector pNBL3 has been constructed by inserting synthetic oligodeoxynucleotides containing a Bacillus ribosome-binding site (RBS) and the ATG codon downstream from dfrA' on pNBL1. When the E. coli lacZ gene was placed downstream from the dfrA' gene in pNBL3, efficient synthesis of beta-galactosidase was observed in both cells, showing that the polycistronic expression system is suitable for directing expression of heterologous genes. Translational efficiency of the lacZ gene on pNBL3 was further examined in B. subtilis by changing the sequence upstream from lacZ. Unlike the results previously reported [Sprengel et al., Nucleic Acids Res. 13 (1985) 893-909], when RBS was present, the high level of lacZ expression was preserved irrespective of spacing between the stop codon of the upstream dfrA' gene and the start codon of the downstream lacZ gene. However, in the absence of RBS, the spacing between both genes affected lacZ expression. That is, translational coupling of dfrA'-lacZ was observed, although the translational efficiency was very low.
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PMID:Construction of a new shuttle expression vector for Bacillus subtilis and Escherichia coli by using a polycistronic system. 314 23

Previous work has shown that a fusion protein bearing a "nonremovable" N-terminal ubiquitin (Ub) moiety is short-lived in vivo, the fusion's Ub functioning as a degradation signal. The proteolytic system involved, termed the UFD pathway (Ub fusion degradation), was dissected in the yeast Saccharomyces cerevisiae by analyzing mutations that perturb the pathway. Two of the five genes thus identified, UFD1 and UFD5, function at post-ubiquitination steps in the UFD pathway. UFD3 plays a role in controlling the concentration of Ub in a cell: ufd3 mutants have greatly reduced levels of free Ub, and the degradation of Ub fusions in these mutants can be restored by overexpressing Ub. UFD2 and UFD4 appear to influence the formation and topology of a multi-Ub chain linked to the fusion's Ub moiety. UFD1, UFD2, and UFD4 encode previously undescribed proteins of 40, 110, and 170 kDa, respectively. The sequence of the last approximately 280 residues of Ufd4p is similar to that of E6AP, a human protein that binds to both the E6 protein of oncogenic papilloma viruses and the tumor suppressor protein p53, whose Ub-dependent degradation involves E6AP. UFD5 is identical to the previously identified SON1, isolated as an extragenic suppressor of sec63 alleles that impair the transport of proteins into the nucleus. UFD5 is essential for activity of both the UFD and N-end rule pathways (the latter system degrades proteins that bear certain N-terminal residues). We also show that a Lys --> Arg conversion at either position 29 or position 48 in the fusion's Ub moiety greatly reduces ubiquitination and degradation of Ub fusions to beta-galactosidase. By contrast, the ubiquitination and degradation of Ub fusions to dihydrofolate reductase are inhibited by the UbR29 but not by the UbR48 moiety. ufd4 mutants are unable to ubiquitinate the fusion's Ub moiety at Lys29, whereas ufd2 mutants are impaired in the ubiquitination at Lys48. These and related findings suggest that Ub-Ub isopeptide bonds in substrate-linked multi-Ub chains involve not only the previously identified Lys48 but also Lys29 of Ub, and that structurally different multi-Ub chains have distinct functions in Ub-dependent protein degradation.
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PMID:A proteolytic pathway that recognizes ubiquitin as a degradation signal. 761 50

A long-standing conundrum of glucocorticoid receptors has been why the steroid binding domain is active in hybrid proteins but not in isolation. For this reason, the precise boundaries of the steroid binding domain have not been defined. These questions have now been systematically examined with a variety of receptor deletion constructs. Plasmids encoding amino acids 537-673 and 537-795 of the rat receptor did not yield stable proteins, while the fusion of receptor or non-receptor sequences upstream of 537-673 afforded stable proteins that did not bind steroid. Wild type steroid binding affinity could be obtained, however, when proteins such as beta-galactosidase or dihydrofolate reductase were fused upstream of receptor amino acids 537-795. Studies of a series of dhfr/receptor constructs with deletions at the amino- and carboxyl-terminal ends of the receptor sequence localized the boundaries of the steroid binding domain to 550-795. The absence of steroid binding upon deletion of sequences in the carboxyl-terminal half of this domain was consistent with improperly folded receptor sequences. This conclusion was supported by analyses of the proteolysis and thermal stability of the mutant receptors. Thus, three independent regions appear to be required for the generation of the steroid binding form of receptors: 1) a protein sequence upstream of the steroid binding domain, which conveys stability to the steroid binding domain, 2) sequences of the carboxyl-terminal amino acids (674-795), which are required for the correct folding of the steroid binding domain, and 3) amino-terminal sequences (550-673), which may be sufficient for steroid binding after the entire steroid binding domain is properly folded. These results establish that the steroid binding domain of glucocorticoid receptors is not independently functional and illustrate the importance of both protein stability and protein folding when constructing mutant proteins.
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PMID:Modular structure of glucocorticoid receptor domains is not equivalent to functional independence. Stability and activity of the steroid binding domain are controlled by sequences in separate domains. 870 25

The hamster polyomavirus (HaPV) is associated with spontaneously appearing skin epithelioma of the Syrian hamster Z3 strain. Virus particles prepared from the skin epithelioma cause lymphoma and leukemia when injected into newborn hamsters from a distinct Syrian hamster colony (HaP); in contrast to the skin epithelioma the hemopoietic tumors are virus free but accumulate viral DNA. To study the humoral immune response of HaPV-infected Z3 hamsters we produced recombinant HaPV proteins in Escherichia coli as beta-galactosidase-, TrpE- and dihydrofolate reductase-fusion proteins or as non-fused proteins. Recombinant plasmids carried segments of all putative early and late HaPV proteins. The recombinant proteins were detected in stained SDS polyacrylamide gels and in Western blots using monoclonal anti-TrpE and anti-beta-galactosidase antibodies and sera of HaPV-infected hamsters. Sera from HaPV-infected Z3 hamsters and crude lysates of all clones were applied to Western blots to characterize the humoral immune response in the animals. HaPV-specific antibodies were found to be directed against early protein segments translated from the first common exon and from the second unique exon of LT and MT, resp., as well as against the late proteins VP1 and VP2/3. The almost complete VP2 was recognized by all sera whereas VP1 was detected only by a half of the sera. Our data suggest the presence of at least 2 immunodominant regions in VP2, one in the C-terminal VP1 and at least 4 in early proteins.
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PMID:Hamster polyomavirus-encoded proteins: gene cloning, heterologous expression and immunoreactivity. 888 64

Type III machines of pathogenic Yersinia spp. transport Yop proteins across the bacterial envelope into host cells. Translational fusions of yopE to the dihydrofolate reductase gene (dhfr) or the beta-galactosidase gene (lacZ) generate hybrid proteins that block type III injection of Yop proteins into host cells, consistent with the canonical view that impassable DHFR and LacZ hybrids jam secretion machines. Mutations in repressors of posttranscriptional gene regulation, Yersinia enterocolitica yscM1 and yscM2 as well as Yersinia pestis lcrQ, relieve the YopE-DHFR-imposed blockade and restore type III injection into host cells. Genetic suppression of the type III blockade does not, however, promote YopE-DHFR secretion. A model is proposed whereby rejection of YopE-DHFR from the secretion pathway inhibits type III gene expression.
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PMID:Rejection of impassable substrates by Yersinia type III secretion machines. 1619 80

Sarcocystis neurona is an apicomplexan parasite that is the major cause of equine protozoal myeloencephalitis (EPM). The biology of this pathogen remains poorly understood in part due to unavailability of molecular genetic tools. Hence, with an objective to develop DNA transfection capabilities for S. neurona, the 5' flanking region of the SnSAG1 gene was isolated from a genomic library and used to construct expression plasmids. In transient assays, the reporter molecules beta-galactosidase (beta-gal) and yellow fluorescent protein (YFP) could be detected in electroporated S. neurona, thereby confirming the feasibility of transgene expression in this organism. Stable transformation of S. neurona was achieved using a mutant dihydrofolate reductase thymidylate synthase (DHFR-TS) gene of Toxoplasma gondii that confers resistance to pyrimethamine. This selection system was used to create transgenic S. neurona that stably express beta-gal and YFP. As shown in this study, these transgenic clones can be useful for analyzing growth rate of parasites in vitro and for assessing drug sensitivities. More importantly, the DNA transfection methods described herein should greatly facilitate studies examining intracellular parasitism by this important coccidian pathogen.
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PMID:Molecular genetic transfection of the coccidian parasite Sarcocystis neurona. 1684 42

Recombinant mammalian cultures for heterologous gene expression typically involve cells traversing the cell cycle. Studies were conducted to characterize rates of accumulation of intracellular foreign protein in single cells during the cell cycle of Chinese hamster ovary (CHO) cells transfected with an expression vector containing the gene for dihydrofolate reductase (dhfr) and the lacZ gene for bacterial beta-galactosidase (a nonsecreated protein). The lacZ gene was under the control of the constitutive cytomegalovirus promoter. These normally attachment-grown cells were adapted to suspension culture in 10(-7) M methotrexate, and a dual-laser flow cytometer was used to simultaneously determine the DNA and foreign protein (beta-galactosidase) content of single living cells. Expression of beta-galactosidase as a function of cell cycle phase was evaluated for cells in the exponential growth phase, early plateau phase, and inhibited traverse of the cell cycle during exponential growth. The results showed that the beta-galactosidase production rate is higher in the S phase than that in the G1 or G2/M phases. Also, when cell cycle progression was stopped at the S phase by addition of aphidicolin, beta-galactosidase content in single cells was higher than that in exponential phase or plateau phase cells and increased with increasing culture time. Although the cells did not continue to divide after aphidicolin addition, the production of beta-galactosidase per unit volume of culture was very similar to that in normal exponential growth.
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PMID:Foreign gene expression (beta-galactosidase) during the cell cycle phases in recombinant CHO cells. 1861 41


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