Biomedical subjects

M V Chao

Publications and source records attributed to M V Chao.

112 records · Page 7Linked to original sources

PubMed1980

lac Operator nucleosomes. 1. Repressor binds specifically to operator within the nucleosome core.

We have shown that the lac repressor can recognize and bind specifically to the lac operator contained in short restriction fragments which have been complexed with the four core histones to form artificial nucleosomes and core particles. These lac reconstitutes have been well characterized, and it is apparent that the operator DNA itself is associated fully and normally with the octameric histone cores. The binding of repressor to these reconstitutes is operator dependent since nucleosomes lacking the operator sequence fail completely to bind repressor under our conditions. Moreover, binding is abolished by IPTG (isopropyl thiogalactoside), further demonstrating operator specificity. Nevertheless, sedimentation studies show that repressor binding does not involve displacement of the histone octamer. Thus, the lac repressor and the histone octamer bind simultaneously to the same DNA. lac reconstitutes, in which the DNA has been cross-linked to the histones with formaldehyde, also support simultaneous specific binding by lac repressor. Since all particles among the reconstitutes, cross-linked or not, bind repressor quantitatively, we infer that the repressor binding surface of the operator DNA always faces generally outward rather than inward toward the histone core. It is likely to be this feature of lac operator particle structure, dictated in an unknown manner by DNA sequence, that allows the simultaneous binding of histones and repressor to the same DNA region.

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PubMed1980

lac Operator nucleosomes. 2. lac Nucleosomes can change conformation to strengthen binding by lac repressor.

We have shown previously that lac repressor binds specifically and quantitatively to lac operator restriction fragments which have been complexed with histones to form artificial nucleosomes (203 base pair restriction fragment) or core particles (144 base pair restriction fragment. We describe here a quantitative method for determining the equilibrium binding affinities of repressor for these lac reconstitutes. Quantitative analysis shows that the operator-histone reconstitutes may be grouped into two affinity classes: those with an affinity for repressor close to that of naked DNA and those with an affinity 2 or more orders of magnitude less than that of naked DNA. All particles in the lac nucleosome preparations bind repressor with high affinity, but the lac core particle preparations contain particles of both high and low affinities for repressor. Formaldehyde cross-linking causes all high-affinity species to suffer a 100-fold decrease in binding affinity. In contrast, there is no effect of cross-linking on species of low affinity. Therefore, the ability of a particle to be bound tightly by repressor depends on a property of the particle which is eliminated by cross-linking. Control experiments have shown that chemical damage to the operator does not accompany cross-linking. Therefore, the property sensitive to cross-linking must be the ability of the particle to change conformation. We infer that the particles of low native affinity, like cross-linked particles, are of low affinity because of an inability to facilitate repressor binding by means of this conformational change. Dimethyl suberimidate cross-linking experiments show that histone-histone cross-linking is sufficient to preclude high-affinity binding. Thus, the necessary conformational change involves a nucleosome histone core event. We find that the ability of a particle to undergo a repressor-induced facilitating conformational change appears to depend on the position of the operator along the DNA binding path of the nucleosome core. We present a general model which proposes that nucleosomes are divided into domains which function differentially to initiate conformational changes in response to physiological stimuli.

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PubMed1979

DNA sequence directs placement of histone cores on restriction fragments during nucleosome formation.

Restriction fragments, 203 and 144 base pairs in length, bearing the Escherichia coli lac control region have been reconstituted with the core histones from calf thymus to form nucleosomes. By several criteria the reconstituted nucleosomes are similar to native nucleosomes obtained by micrococcal nuclease digestion of calf thymus nuclei. However, sensitive nuclease digestion studies reveal subtle and important differences between native monosomes and the lac reconstitutes. Each reconstitute consists mainly of nucleosomes containing histone cores placed nonrandomly with respect to the DNA sequence. The shorter reconstitute forms asymmetric nucleosomes as evidenced by the DNase I digestion pattern. Exonuclease III digestion followed by 5'-end analysis of the larger reconstitute suggests that, of the many possible arrangements of histone core with DNA sequence, only two are highly favored.

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PubMedDate not supplied

p75 neurotrophin receptor as a modulator of survival and death decisions.

The p75 receptor is the founding member of the TNF receptor superfamily. Members in this receptor family share a common cysteine motif repeated two to six times that serves as the ligand binding domain. In addition, several members contain a cytoplasmic region designated the death domain. The neurotrophins NGF, BDNF, NT-3, and NT-4 each bind to the p75 receptor and also more selectively to members of the Trk family of receptor tyrosine kinases. Although the biological functions of p75 have been elusive, recent experimental evidence supports an involvement of this receptor in apoptosis. This presents a counter-intuitive function for neurotrophins, which are normally required for the survival of neurons during development. The life-and-death decisions by neurotrophins appear to be governed by the level of expression and signaling activities of the p75 and Trk tyrosine kinase receptors and their downstream effector molecules. The generation of the correct number of cells in the nervous system is a highly controlled and coordinated process that is the consequence of cell proliferation and cell death decisions. The appropriate number of neuronal and glial cells formed during development guarantees the establishment of proper innervation and functional synaptic connections. One common mechanism to account for the number of viable cells is the ability to form ligand-receptor complexes that promote cell survival under conditions of limiting concentrations of trophic factors. Another diametrically opposed mechanism is to produce ligand-receptor interactions that can activate programmed cell death directly.

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