Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.2.1.23 (beta-galactosidase)
 14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have constructed a total deletion of the regulatory gene LEU3. Comparing the deletion mutant with a leu3 spontaneous mutant, we find that both types of mutants have lost the ability to regulate a LEU2'-lacZ translational fusion by the LEU3-alpha-isopropylmalate-dependent mechanism, which we confirm to be the major regulatory mechanism for LEU2. Surprisingly, cells containing the total leu3 deletion are more leaky (i.e. grow better in the absence of extraneous leucine) than cells containing a spontaneous leu3 mutation. Accompanying the growth rate difference is a difference in the expression of the LEU2-lacZ fusion: the specific activity of beta-galactosidase amounts to about 8% of a wild type control in a leu3 total deletion mutant, but drops to about 2% in a leu3 spontaneous mutant. The spontaneous mutant differs from the total deletion mutant in that it produces an inactive protein which is still able to bind to the LEU2 upstream activating sequence. We conclude that a basal level control of LEU2 becomes manifest in the absence of LEU3 and is interfered with when LEU3 protein binds to the LEU2 promoter. This conclusion is supported by the finding that a mutant which contains an intact LEU3 gene but is unable to generate alpha-isopropylmalate also interferes with basal level expression of LEU2. Basal level expression depends upon the GCN4 protein, even though LEU2 is not subject to derepression by the general amino acid control system. Changes in the steady-state concentration of LEU2 mRNA show the same trend as changes in the specific activity of the LEU2-lacZ fusion protein, suggesting that regulation of LEU2 expression at both the basal and nonbasal levels is largely transcriptional. The role of alpha-isopropylmalate in the regulation of LEU2 expression appears to be that of a co-activator. Employing mobility shift assays, we show that specific interaction between the LEU3 protein and a 30-base pair DNA fragment carrying the upstream activating sequence of LEU2 takes place irrespective of the presence or absence of alpha-isopropylmalate.
 ...
PMID:Regulation of yeast LEU2. Total deletion of regulatory gene LEU3 unmasks GCN4-dependent basal level expression of LEU2. 219 25

We have constructed a derivative of the bacteriophage Mu (called MudIIZZ1), which contains the lacZ gene coding for beta-galactosidase (beta Gal) and markers suited for yeast transformation (2 mu circle replication origin and LEU2). This new transposon is an efficient tool for studying the expression of cloned yeast nucleotide sequences through beta Gal-protein fusions. It is also adapted for one-step disruption experiments so that a functional map of the same sequence can be drawn. We have used this MudIIZZ1 transposon to study a 5-kb DNA fragment which had been cloned by complementation of a cold-sensitive respiration-deficient phenotype. By testing the expression of the beta Gal fusions and the disruption phenotype, we have confirmed the presence of a gene required for mitochondrial functions, and revealed another two open reading frames in the same fragment; one of these also interferes with mitochondrial biogenesis. The method is fast and reliable, and has potential for more general purposes which are discussed.
 ...
PMID:In vivo functional characterization of a yeast nucleotide sequence: construction of a mini-Mu derivative adapted to yeast. 283 69

We report yeast/Escherichia coli shuttle vectors suitable for fusing yeast promoter and coding sequences to the lacZ gene of E. coli. The vectors contain a region of multiple unique restriction sites including EcoRI, KpnI, SmaI, BamHI, XbaI, SalI, PstI, SphI and HindIII. The region with the unique cloning sites has been introduced in both orientations with respect to lacZ and occurs proximal to the eighth codon of the gene. All the restriction sites have been phased to three different reading frames. Two series of vectors have been constructed. The first series (YEp) has two origins of replication (ori), i.e., of the yeast 2 mu circle and of the ColE1 plasmid of E. coli, and can therefore replicate autonomously in both organisms. These shuttle vectors also have the ApR gene of E. coli and either the yeast LEU2 or URA3 genes to allow for selection of both E. coli and yeast transformants. The second series of vectors (YIp) are identical in all respects to the YEp vectors except that they lack the 2 mu ori. The YIp vectors can be used to integrate lacZ fusions into yeast chromosomal DNA. None of the vectors express beta-galactosidase (beta Gal) in yeast or E. coli in the absence of inserted yeast promoter sequences. The 5'-nontranslated sequences and parts of the coding sequences of various yeast genes have been cloned into representative lacZ fusion vectors. In-frame gene fusions can be detected by beta Gal activity when either yeast or E. coli clones are plated on media containing XGal indicator. Quantitative determinations of promoter activity were made by colorimetric assay of beta Gal activity in whole cells. Fusion of the yeast CYC1 gene to lacZ in one of the vectors allowed detection of regulated expression of this gene when cells were grown under conditions of catabolite repression or derepression.
 ...
PMID:Yeast shuttle and integrative vectors with multiple cloning sites suitable for construction of lacZ fusions. 302 15

Two yeast/E. coli shuttle vectors have been constructed. The two vectors, YEp351 and YEp352, have the following properties: (1) they can replicate autonomously in Saccharomyces cerevisiae and in E. coli; (2) they contain the beta-lactamase gene and confer ampicillin resistance to E. coli; (3) they contain the entire sequence of pUC18; (4) all ten restriction sites of the multiple cloning region of pUC18 including EcoRI, SacI, KpnI, SmaI, BamHI, XbaI, SalI, PstI, SphI and HindIII are unique in YEp352; these sites are also unique in YEp351 except for EcoRI and KpnI, which occur twice; (5) recombinant plasmids with DNA inserts in the multiple cloning region of YEp351 and YEp352 can be recognised by loss of beta-galactosidase function in appropriate E. coli hosts; (6) YEp351 and YEp352 contain the yeast LEU2 and URA3 genes, respectively, allowing for selection of these auxotrophic markers in yeast and E. coli; (7) both plasmids are retained with high frequency in yeast grown under non-selective conditions indicative of high plasmid copy number. The above properties make the shuttle vectors suitable for construction of yeast genomic libraries and for cloning of DNA fragments defined by a large number of different restriction sites. The two vectors have been further modified by deletion of the sequences necessary for autonomous replication in yeast. The derivative plasmids YIp351 and YIp352 can therefore be used to integrate specific sequences into yeast chromosomal DNA.
 ...
PMID:Yeast/E. coli shuttle vectors with multiple unique restriction sites. 333 5

The SUC2 gene produces two differently regulated mRNAs that encode two forms of invertase. The 1.9-kilobase mRNA encoding secreted invertase is regulated by glucose (carbon catabolite) repression, and the 1.8-kilobase mRNA encoding intracellular invertase is synthesized constitutively. Previous work has shown that the 5' noncoding region between -650 and -418 is required for derepression of secreted invertase in response to glucose deprivation. We show here that this upstream region can confer glucose-repressible expression to a heterologous gene, a LEU2-lacZ gene fusion, that is not normally regulated by glucose repression. This expression was found to respond appropriately to mutations in trans-acting genes that affect regulation of SUC2 expression. Mutations in the SNF1 through SNF6 loci reduced derepression of beta-galactosidase, and a mutation at the SSN6 locus caused constitutive expression. These findings indicate that the SUC2 upstream region mediates the regulatory effects of these genes and suggest that regulation occurs at the level of transcription. In addition, the upstream region was partially active in the inverted orientation.
 ...
PMID:Upstream region of the SUC2 gene confers regulated expression to a heterologous gene in Saccharomyces cerevisiae. 393 53

The interactions between CD4 or CD8 and p56lck were tested using the two-hybrid protein interaction system in yeast. Plasmid constructs were created which fuse the cytoplasmic domains of either CD4 or CD8 alpha to the DNA-binding protein LexA, and the unique amino-terminal domain of p56lck fused to a transcriptional activation domain. These constructs were transfected into yeast bearing lacZ and LEU2 reporter genes controlled by upstream LexA operator sequences. Yeast transfectants bearing either CD4 or CD8 alpha hybrid proteins in combination with the amino terminal p56lck hybrid protein exhibited increased beta-galactosidase activity and growth on leucine-deficient medium, indicating interactions between these protein domains. Quantitation of reporter activation indicated that the interaction of p56lck with CD8 alpha is at least 18-fold weaker than the interaction with CD4 in this assay. This reduced interactive capacity is apparently not due to competition by CD8 alpha interacting with itself, since homotypic or heterotypic interactions between CD8 alpha and/or CD4 could not be detected. Truncation and point mutants demonstrated that the interactions of p56lck with CD4 or CD8 alpha were dependent on the integrity of a pair of cysteines on each protein. The results indicate that these interactions do not require any additional proteins. Additionally, expression of the entire p56lck molecule as a hybrid with LexA resulted in dramatic reduction in the growth of yeast. Though the two-hybrid system is a powerful tool for examining protein interactions, this result indicates potential limitations in studying full-length src family tyrosine kinases in yeast.
 ...
PMID:Interactions between the amino-terminal domain of p56lck and cytoplasmic domains of CD4 and CD8 alpha in yeast. 766 3

The Saccharomyces cerevisiae CRY1 gene encodes the 40S ribosomal subunit protein rp59 and confers sensitivity to the protein synthesis inhibitor cryptopleurine. A yeast strain containing the cry1-delta 1::URA3 null allele is viable, cryptopleurine sensitive (CryS), and expresses rp59 mRNA, suggesting that there is a second functional CRY gene. The CRY2 gene has been isolated from a yeast genomic library cloned in bacteriophage lambda, using a CRY1 DNA probe. The DNA sequence of the CRY2 gene contains an open reading frame encoding ribosomal protein 59 that differs at five residues from rp59 encoded by the CRY1 gene. The CRY2 gene was mapped to the left arm of chromosome X, centromere-proximal to cdc6 and immediately adjacent to ribosomal protein genes RPS24A and RPL46. Ribosomal protein 59 is an essential protein; upon sporulation of a diploid doubly heterozygous for cry1-delta 2::TRP1 cry2-delta 1::LEU2 null alleles, no spore clones containing both null alleles were recovered. Several results indicate that CRY2 is expressed, but at lower levels than CRY1: (1) Introduction of CRY2 on high copy plasmids into CryR yeast of genotype cry1 CRY2 confers a CryS phenotype. Transformation of these CryR yeast with CRY2 on a low copy CEN plasmid does not confer a CryS phenotype. (2) Haploids containing the cry1-delta 2::TRP1 null allele have a deficit of 40S ribosomal subunits, but cry2-delta 1::LEU2 strains have wild-type amounts of 40S ribosomal subunits. (3) CRY2 mRNA is present at lower levels than CRY1 mRNA. (4) Higher levels of beta-galactosidase are expressed from a CRY1-lacZ gene fusion than from a CRY2-lacZ gene fusion. Mutations that alter or eliminate the last amino acid of rp59 encoded by either CRY1 or CRY2 result in resistance to cryptopleurine. Because CRY2 (and cry2) is expressed at lower levels than CRY1 (and cry1), the CryR phenotype of cry2 mutants is only expressed in strains containing a cry1-delta null allele.
 ...
PMID:Molecular genetics of cryptopleurine resistance in Saccharomyces cerevisiae: expression of a ribosomal protein gene family. 829 76

The kinetics of amyloid fibril formation by beta-amyloid peptide (Abeta) are typical of a nucleation-dependent polymerization mechanism. This type of mechanism suggests that the study of the interaction of Abeta with itself can provide some valuable insights into Alzheimer disease amyloidosis. Interaction of Abeta with itself was explored with the yeast two-hybrid system. Fusion proteins were created by linking the Abeta fragment to a LexA DNA-binding domain (bait) and also to a B42 transactivation domain (prey). Protein-protein interactions were measured by expression of these fusion proteins in Saccharomyces cerevisiae harboring lacZ (beta-galactosidase) and LEU2 (leucine utilization) genes under the control of LexA-dependent operators. This approach suggests that the Abeta molecule is capable of interacting with itself in vivo in the yeast cell nucleus. LexA protein fused to the Drosophila protein bicoid (LexA-bicoid) failed to interact with the B42 fragment fused to Abeta, indicating that the observed Abeta-Abeta interaction was specific. Specificity was further shown by the finding that no significant interaction was observed in yeast expressing LexA-Abeta bait when the B42 transactivation domain was fused to an Abeta fragment with Phe-Phe at residues 19 and 20 replaced by Thr-Thr (AbetaTT), a finding that is consistent with in vitro observations made by others. Moreover, when a peptide fragment bearing this substitution was mixed with native Abeta-(1-40), it inhibited formation of fibrils in vitro as examined by electron microscopy. The findings presented in this paper suggest that the two-hybrid system can be used to study the interaction of Abeta monomers and to define the peptide sequences that may be important in nucleation-dependent aggregation.
 ...
PMID:Two-hybrid system as a model to study the interaction of beta-amyloid peptide monomers. 870 Aug 86

A single copy of a reporter gene cassette, such as PGKP-lacZ-LEU2 (promoter-reporter-marker gene) cassette, was inserted into one of 32 positions along chromosome III in Saccharomyces cerevisiae with an interval of approximately 10 kb. The amounts of translational gene product (beta-galactosidase) synthesized by the cassette-transformed cells were then determined. The region specificity in chromosome III could be demonstrated in gene expression: two higher-expressed regions (hot regions), 133 and 199 (MAT) regions, and seven lower-expressed regions (cold regions). For the steady and high production of polypeptide, foreign gene products, by yeast, we would like to state that we hope for an insertion of the artificially prepared gene cassette [(promoter)-(foreign gene)-(marker gene) ] into a hot region, such as 199 (MAT) region of chromosome III.
 ...
PMID:Region specificity of chromosome III on gene expression in the yeast Saccharomyces cerevisiae. 1250 22