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lacz

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Official Full Name
Beta galactosidase
Background
Beta galactosidase is coded by a gene (lac z) in the lac operon of Escherichia coli. It is a metalloenzyme that splits lactose into glucose and galactose. It hydrolyzes terminal, non-reducing beta-D-galactose residues in beta-D-galactosides.
Synonyms
LacZ; beta-D-galactosidase; ECK0341; JW0335

Recent Progress

It is generally acknowledged that LacZ has two functional activities. It is capable of hydrolyzing lactose to galactose and glucose and catalyzing the intramolecular isomerization of lactose to allolactose(Fig. 1).

Fig. 1. Stereoviews of LacZ with allolactose, Bis-Tris, and l-ribose ligands. (RW Wheatley et al, 2013)

In 103 muscle cells of the nematode Caenorhabditis elegans, the product of an integrated transgene offers a convenient and cell-specific reporter of intracellular protein catabolism. The transgene is essentially a fusion of a 5'-region of the C. elegans unc-54 gene to the lacZ gene of Escherichia coli. The transgene encodes a 146-kDa fusion polypeptide that in turn forms active beta-galactosidase tetramers. In vivo, this particular protein is stable in well-fed animals, however, after removal of the food source, it is inactivated exponentially with an initial lag of 8 h. From the data collected, it can be concluded that degradation is paradoxically faster than inactivation. Moreover, several characteristic immune-reactive degradation intermediates are also found in active protein, part of which conjugated to ubiquitin. These findings, taken together, proposed that the initial proteolytic cleavages occur in the cytosol, highly likely through an ubiquitin-mediated pathway.

To thoroughly understand the cellular physiology and the regulation of the AOX1 promoter, also to quantitatively evaluate the benefits of a methanol/sorbitol co-feeding process in which the pAOX1-lacZ construct was used as a reporter gene, researchers performed a series of transient continuous cultures. The findings revealed quantitative insight into the co-feeding process, also provided data for the control of methanol/sorbitol co-feeding. Given the data collected, the investigators believed that the productivity of lacZ could be improved about 40% with the optimally mixed feed.

Based on G794A-β-galactosidase (a substituted enzyme) trapping the allolactose, researchers present structural data that maps the glucose site. Further investigation suggested that the glucose from the trapped allolactose is in the acceptor position. A loop covering the Ser-796 and Glu-797 closes over the active site. This loop seems important for the two functional activities of LacZ, since it helps shape the glucose binding site. More intriguingly, given the mobility of the loop, glucose binding becomes transient, thus allowing the release of some glucose. Further bioinformatics studies and genomic analyses indicated that the glucose binding site of LacZ played a crucial role in evolution of lac operon.

β-Propiolactone (BPL) and N -methyl- N ′-nitro- N -nitrosoguanidine (MNNG) are two direct alkylating agents. They are capable of inducing multiple genetic lesions and tumors in the rodent stomach. In order to determine the optimal sampling time and also to investigate the cause-effect relationship between DNA damage and gene mutations, researchers measured the kinetics of the induction of DNA damage and the induction of gene mutations. After a single oral administration of BPL or MNNG given at certain time, the lacZ mutant frequency was strongly enhanced at the highest doses and all sampling times. Only a small increase in lac Z mutant frequency was found at the lowest doses and both sampling times. These findings, taken together indicated the ability and complementarity of the SCGE and Muta64Mouse models to assess the genotoxicity of direct alkylating agents in the mouse gastric mucosa in vivo.

Through determination of the nucleotide sequence of the Escherichia coli/Aggregatibacter actinomycetemcomitans shuttle vector pYGK, researchers were allowed to design and construct a new shuttle cloning vector, pJT4, and promoterless lacZ transcriptional/translational fusion plasmids, pJT3 and pJT5. More specifically, plasmids pJT4 and pJT5 possess the origin of replication needed to maintain shuttle vector replication. moreover, to generate a scarless and markerless deletion mutations of genes in the oral pathogen A. actinomycetemcomitans, researchers also designed a new suicide vector pJT1. Plasmid pJT1 does not leave antibiotic markers or scars on the chromosome even after gene deletion, thus allow the researchers to combine several mutations in the same genetic background. 

References:

  1. Zdinak, L. A., et al. "Transgene-coded chimeric proteins as reporters of intracellular proteolysis: starvation-induced catabolism of a lacZ fusion protein in muscle cells of Caenorhabditis elegans." Journal of Cellular Biochemistry 67.1(2015):143-153.
  2. Niu, Hongxing, et al. "A quantitative study of methanol/sorbitol co-feeding process of a Pichia pastoris Mut+/pAOX1-lacZ strain." Microbial Cell Factories 12.1(2013):33.
  3. Wheatley, R. W., et al. "Structural explanation for allolactose (lac operon inducer) synthesis by lacZ β-galactosidase and the evolutionary relationship between allolactose synthesis and the lac repressor. " Journal of Biological Chemistry 288.18(2013):12993-13005.
  4. Brault, D, et al. "Kinetics of induction of DNA damage and lacZ, gene mutations in stomach mucosa of mice treated with β-propiolactone and N -methyl- N ′-nitro- N -nitrosoguanidine, using single-cell gel electrophoresis and Muta TM Mouse models." Environmental & Molecular Mutagenesis 34.2‐3(2015):182-189.
  5. Mar&xed, et al. "Construction of new cloning, lacZ reporter and scarless-markerless suicide vectors for genetic studies in Aggregatibacter actinomycetemcomitans." Plasmid 69.3(2013):211-222.