Analysis of RNA-DNA competition hybridization using the Scatchard plot

The Scatchard plot is convenient for expressing RNA-DNA competition hybridization data because the maximum amount of competition is directly read as the X-intercept. The derivation which validates the use of this graphical method for competition hybridization data is presented.     

DNA-RNA hybridization.

Interest in nucleic acid hybridization stems mainly from its great power as a tool in biological research. It is used in several quite distinct ways. Because of the high degree of specificity that they show, hybridization techniques can be used to measure the amount of one specific sequence within a very heterogeneous mixture of sequences. Measurements of 1/10(6)-10(7) have been recorded. In extension of this, various properties of a specific sequence can often be studied. Secondly, because the kinetics of nucleic acid hybridization are quite well understood, it can be used to characterize both a pure sequence and a very complex mixture of sequences, like the genome of a vertebrate. Thirdly, again because of its specificity, it can be used to measure homologies between different populations of nucleic acids. Lastly, in conjunction with other techniques, it can be used as a basis for the fractionation of nucleic acid populations and the purification of specific sequences. Specific examples of these applications are given, with special reference to the organization of the genome in higher eukaryotes.     

In situ DNA-RNA hybridization using in vivo bromodeoxyuridine-labeled DNA probe

Summary An in vivo 5-bromodeoxyuridine (BrdUrd) labeled DNA probe was used for in situ DNA-RNA hybridization. BrdUrd was incorporated into plasmid DNA by inoculating E. coli with Luria-Bertani (LB) culture medium containing 500 mg/L of BrdUrd. After purification of the plasmid DNA, specific probes of the defined DNA fragments, which contained the cloned insert and short stretches of the vector DNA, were generated by restriction endonuclease. The enzymatic digestion pattern of the BrdUrd-labeled plasmid DNA was the same as that of the non-labeled one. BrdUrd was incorporated in 15%–20% of the total DNA, that is, about 80% of the thymidine was replaced by BrdUrd. Picogram amounts of the BrdUrd-labeled DNA probe itself and the target DNA were detectable on nitrocellulose filters in dot-blot spot and hybridization experiments using a peroxidase/diaminobenzidine combination. The BrdUrd-labeled DNA probe was efficiently hybridized with both single stranded DNA on nitrocellulose filters and cellular mRNA in in situ hybridization experiments. Through the reaction with BrdUrd in single stranded tails, hybridized probes were clearly detectable with fluorescent microscopy using a FITC-conjugated monoclonal anti-BrdUrd antibody. The in vivo labeling method did not require nick translation steps or in vitro DNA polymerase reactions. Sensitive, stable and efficient DNA probes were easily obtainable with this method.     

In situ DNA-RNA hybridization using in vivo bromodeoxyuridine-labeled DNA probe

An in vivo 5'-bromodeoxyuridine (BrdUrd) labeled DNA probe was used for in situ DNA-RNA hybridization. BrdUrd was incorporated into plasmid DNA by inoculating E. coli with Luria-Bertani (LB) culture medium containing 500 mg/L of BrdUrd. After purification of the plasmid DNA, specific probes of the defined DNA fragments, which contained the cloned insert and short stretches of the vector DNA, were generated by restriction endonuclease. The enzymatic digestion pattern of the BrdUrd-labeled plasmid DNA was the same as that of the non-labeled one. BrdUrd was incorporated in 15%-20% of the total DNA, that is, about 80% of the thymidine was replaced by BrdUrd. Picogram amounts of the BrdUrd-labeled DNA probe itself and the target DNA were detectable on nitrocellulose filters in dot-blot spot and hybridization experiments using a peroxidase/diaminobenzidine combination. The BrdUrd-labeled DNA probe was efficiently hybridized with both single stranded DNA on nitrocellulose filters and cellular mRNA in in situ hybridization experiments. Through the reaction with BrdUrd in single stranded tails, hybridized probes were clearly detectable with fluorescent microscopy using a FITC-conjugated monoclonal anti-BrdUrd antibody. The in vivo labeling method did not require nick translation steps or in vitro DNA polymerase reactions. Sensitive, stable and efficient DNA probes were easily obtainable with this method.     

Linearization of two ligand-one binding site scatchard plot and the "IC50" competitive inhibition plot: application to the simplified graphical determination of equilibrium constants

A simple procedure for linearizing the curved, two ligand-one binding site Scatchard plot resulting from the presence of a constant concentration of competitive inhibitor is proposed; the same procedure may also be applied to the analysis of data derived from the "IC50" competitive inhibition experimental design. Furthermore, a useful generalization of the Cheng-Prusoff correction is presented.     

Studies by DNA-RNA hybridization of transcriptional diversity in human brain

Abstract— Purified unique sequences of human DNA (from HeLa cells) were hybridized with various preparations of human brain KNA, to obtain estimates of the fraction of DNA transcribed. Values of 12 per cent were obtained for fetal brain increasing to 24 per cent for specific regions of adult brain. Right and left parietal and temporal cortical RNA gave similar plateau values but left frontal cortical RNA appeared to represent 2–3 times as much of the total genome as that from right frontal cortex. Lower values were obtained for fetal and adult brain stem and for RNA prepared from temporal cortex of a patient exhibiting gross cerebral atrophy. Attempts were made to assess the contributions of glial and neuronal cells by using human astrocytoma and mouse neuroblastoma RNA.     

Heterogeneities in EGF receptor density at the cell surface can lead to concave up scatchard plot of EGF binding

The mechanism responsible for the concave up nature of the Scatchard plot of epidermal growth factor (EGF) binding on EGF receptor (EGFR) has been a controversial issue for more than a decade. Past efforts to mechanistically simulate the concave up nature of the Scatchard plot of EGF binding have shown that negative cooperativity in EGF binding on an EGFR dimer or inclusion of some external site or binding event can describe this behavior. However, herein we show that heterogeneity in the density of EGFR due to localization in certain regions of the plasma membrane, which has been experimentally reported, can also lead to concave up shape of the Scatchard plot of the EGF binding on EGFR.     

Dual labeling in standard DNA-RNA hybridization studies using 125 I-labeled nuclear RNA and 3H-labeled DNA.

Standard DNA-RNA hybridization studies, using nucleic acids isolated from mammalian tissues, are frequently hindered by relatively low levels of radioactivity in pulse-labeled RNA and in an inability to reliably estimate the amount of DNA present in the hybrid. In the method described here nuclear RNA is labeled in vitro with 125I to 400 000- 800 000 cpm/mug and DNA is obtained from a rat glial tumor line grown in culture and labeled to specific activities of 42 000-79 000 cpm/mug. DNA-RNA hybridization is conducted in an all solution system at RNA:DNA ratios of 3.5:1 to 18:1. Assay background is controlled by pretreatment of the hybrid and free RNA at the conclusion of the annealing study with RNase, then isolation of the hybrid together with a small fraction of free RNA oligonucleotides on hydroxyapatite. The partially purified hybrids are then trapped on Millipore filters. Assay background id 0.004% of total counts present in the annealing reaction. Comparison of the annealing reactions of pulse-labeled liver nuclear RNA and in vitro 125I-labeled nuclear RNA in saturation, kinetic, and competitive hybridization studies shows them to be essentially the same. Nuclear RNA labeled by either tritium or iodine shows a 10-20-fold greater concentration of the annealing sequences over that found in the microsomal RNA. Minor differences are noted between the nuclear RNAs in the initial rates of reaction and in the magnitude of the decrease in percent hybridization at low levels of unlabeled competitor RNA. This may be due to preferential labeling in pulse-labeled RNA of molecules which are present in lower concentrations or are transcribed from more frequently repeated DNA sequences than the average population of annealing RNA molecules. The technique has application in systems where the amount of tissue for RNA extraction is small or where the system does not permit the obtaining of pulse-labeled RNA, as in experimental rodent skin carcinogenesis or in dealing with RNA from the tissues of large mammals or humans.     

Physical mapping of complex mRNA populations by "criss-cross" DNA-RNA hybridization

The “criss-cross” hybridization technique, originally developed to construct restriction enzyme-generated linkage maps of DNA was adapted to allow simulataneous size estimates of mRNAs, and their location on such physical maps. The technique consists of blot transferring a ³²P-labeled, gel-fractionated mRNA population to a nitrocellulose filter to which a restriction digest of DNA has previously been blot transferred. The RNA transfer is performed under hybridization conditions and perpendicular to the axis of the DNA pattern. The width of the bands in the DNA and RNA gels are controlled such that the resulting matrix allows every mRNA species to cross every restriction fragment band. Thus whenever an mRNA band intersects a DNA band containing complementary sequences, hybridization can occur, and be detected by autoradiography. Each spot in the resulting pattern has size and map location characteristics determined by the electrophoretic mobility of the mRNA band (relative to ribosomal RNA markers) and the physical coordinates of the DNA fragment on the restriction map. As an example of the technique, at least 12 of the late mRNAs of adenovirus type 2 were located on the SmaI physical map of the 35 kbp genome of the virus. In addition, the transciption orientation of mRNAs was determined by hybridization to separated strands of the BamHI fragments.