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Lactase is an enzyme produced by many organisms and is essential to the complete digestion of whole milk. It breaks down the sugar lactose into its component parts, galactose and glucose. Lactase is found in the brush border of the small intestine of humans and other mammals. People deficient in lactase or lacking functional lactase may experience the symptoms of lactose intolerance after consuming milk products. Microbial β-galactosidase can be purchased as a food supplement and is added to milk to produce "lactose-free" milk products.
Galactose, sometimes abbreviated Gal, is a monosaccharide sugar that is about as sweet as glucose, and about 65% as sweet as sucrose. It is an aldohexose and a C-4 epimer of glucose. A galactose molecule linked with a glucose molecule forms a lactose molecule.
β-Galactosidase is a glycoside hydrolase enzyme that catalyzes hydrolysis of terminal non-reducing β-D-galactose residues in β-D-galactosides.
François Jacob was a French biologist who, together with Jacques Monod, originated the idea that control of enzyme levels in all cells occurs through regulation of transcription. He shared the 1965 Nobel Prize in Medicine with Jacques Monod and André Lwoff.
A cloning vector is a small piece of DNA that can be stably maintained in an organism, and into which a foreign DNA fragment can be inserted for cloning purposes. The cloning vector may be DNA taken from a virus, the cell of a higher organism, or it may be the plasmid of a bacterium. The vector contains features that allow for the convenient insertion of a DNA fragment into the vector or its removal from the vector, for example through the presence of restriction sites. The vector and the foreign DNA may be treated with a restriction enzyme that cuts the DNA, and DNA fragments thus generated contain either blunt ends or overhangs known as sticky ends, and vector DNA and foreign DNA with compatible ends can then be joined by molecular ligation. After a DNA fragment has been cloned into a cloning vector, it may be further subcloned into another vector designed for more specific use.
The lactose operon is an operon required for the transport and metabolism of lactose in E. coli and many other enteric bacteria. Although glucose is the preferred carbon source for most enteric bacteria, the lac operon allows for the effective digestion of lactose when glucose is not available through the activity of β-galactosidase. Gene regulation of the lac operon was the first genetic regulatory mechanism to be understood clearly, so it has become a foremost example of prokaryotic gene regulation. It is often discussed in introductory molecular and cellular biology classes for this reason. This lactose metabolism system was used by François Jacob and Jacques Monod to determine how a biological cell knows which enzyme to synthesize. Their work on the lac operon won them the Nobel Prize in Physiology in 1965.
Isopropyl β-d-1-thiogalactopyranoside (IPTG) is a molecular biology reagent. This compound is a molecular mimic of allolactose, a lactose metabolite that triggers transcription of the lac operon, and it is therefore used to induce protein expression where the gene is under the control of the lac operator.
α-Galactosidase is a glycoside hydrolase enzyme that catalyses the following reaction:
X-gal is an organic compound consisting of galactose linked to a substituted indole. The compound was synthesized by Jerome Horwitz and collaborators in 1964. The formal chemical name is often shortened to less accurate but also less cumbersome phrases such as bromochloroindoxyl galactoside. The X from indoxyl may be the source of the X in the X-gal contraction. X-gal is often used in molecular biology to test for the presence of an enzyme, β-galactosidase, in the place of its usual target, a β-galactoside. It is also used to detect activity of this enzyme in histochemistry and bacteriology. X-gal is one of many indoxyl glycosides and esters that yield insoluble blue compounds similar to indigo dye as a result of enzyme-catalyzed hydrolysis.
The blue–white screen is a screening technique that allows for the rapid and convenient detection of recombinant bacteria in vector-based molecular cloning experiments. This method of screening is usually performed using a suitable bacterial strain, but other organisms such as yeast may also be used. DNA of transformation is ligated into a vector. The vector is then inserted into a competent host cell viable for transformation, which are then grown in the presence of X-gal. Cells transformed with vectors containing recombinant DNA will produce white colonies; cells transformed with non-recombinant plasmids grow into blue colonies.
In molecular cloning, a vector is any particle used as a vehicle to artificially carry a foreign nucleic sequence – usually DNA – into another cell, where it can be replicated and/or expressed. A vector containing foreign DNA is termed recombinant DNA. The four major types of vectors are plasmids, viral vectors, cosmids, and artificial chromosomes. Of these, the most commonly used vectors are plasmids. Common to all engineered vectors are an origin of replication, a multicloning site, and a selectable marker.
pUC19 is one of a series of plasmid cloning vectors designed by Joachim Messing and co-workers. The designation "pUC" is derived from the classical "p" prefix and the abbreviation for the University of California, where early work on the plasmid series had been conducted. The pUC plasmids are all circular double stranded DNA about 2700 base pairs in length. The pUC plasmids are some of the most widely used cloning vectors. This is in part because cells that have successfully been transformed can be easily distinguished from those that have not based on color differences of colonies. pUC18 is similar to pUC19, but the MCS region is reversed.
Lactose permease is a membrane protein which is a member of the major facilitator superfamily. Lactose permease can be classified as a symporter, which uses the proton gradient towards the cell to transport β-galactosides such as lactose in the same direction into the cell.
Galactosidase alpha is an enzyme that in humans is encoded by the GLA gene.
Molecular cloning is a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules and to direct their replication within host organisms. The use of the word cloning refers to the fact that the method involves the replication of one molecule to produce a population of cells with identical DNA molecules. Molecular cloning generally uses DNA sequences from two different organisms: the species that is the source of the DNA to be cloned, and the species that will serve as the living host for replication of the recombinant DNA. Molecular cloning methods are central to many contemporary areas of modern biology and medicine.
In molecular biology, glycoside hydrolase family 27 is a family of glycoside hydrolases.
In molecular biology, glycoside hydrolase family 36 is a family of glycoside hydrolases.
For pharmacology and genetics, the Umu Chromotest, first developed and published by Oda et al., is a biological assay (bioassay) to assess the genotoxic potential of chemical compounds. It is based on the ability of DNA-damaging agents to induce the expression of the umu operon. In connection with the damage inducible (din) genes recA, lexA and umuD, the umuC gene is essentially involved in bacterial mutagenesis through the SOS response.
The CcdA/CcdB Type II Toxin-antitoxin system is one example of the bacterial toxin-antitoxin (TA) systems that encode two proteins, one a potent inhibitor of cell proliferation (toxin) and the other its specific antidote (antitoxin). These systems preferentially guarantee growth of plasmid-carrying daughter cells in a bacterial population by killing newborn bacteria that have not inherited a plasmid copy at cell division.
Marcos Boris Rotman was a Chilean American immunologist–molecular biologist and professor emeritus of Medical Science at Alpert Medical School of Brown University. He is widely recognized for performing the first single molecule experiments in biology. He died in July 2021 at the age of 96.
References
- 1 2 King RC, Mulligan PK, Stansfield WD (2013). A Dictionary of Genetics (8th ed.). Oxford University Press. ISBN 978-0-19-937686-5.
- ↑ Kint JA (February 1970). "Fabry's disease: alpha-galactosidase deficiency". Science. 167 (3922): 1268–1269. Bibcode:1970Sci...167.1268K. doi:10.1126/science.167.3922.1268. PMID 5411915.
- ↑ Broome AM, Bhavsar N, Ramamurthy G, Newton G, Basilion JP (February 2010). "Expanding the utility of beta-galactosidase complementation: piece by piece". Molecular Pharmaceutics. 7 (1): 60–74. doi:10.1021/mp900188e. PMC 2835542 . PMID 19899815.
- ↑ Clark D, Pazdernik N (2013). Molecular Biology. Oxford, UK: Elsevier. p. 201. ISBN 978-0-12-378594-7.