Non-nuclear Proteins Research Articles

Binding of the high mobility group protein, H6, to trout testis chromatin.

When 125I-labeled H6 was incubated with trout testis nuclei under conditions of pH and ionic strength approximating those in vivo, the radioactivity bound nearly quantitatively to the chromatin. Under comparable conditions, most non-nuclear proteins do not bind. Binding was neither tissue- nor species-specific, and HMG-17, a mammalian homolog of H6, behaved similarly to H6. The labeled and the endogenous H6 molecules were equivalent by several criteria: 1) Both were released nearly quantitatively upon treatment of the chromatin with DNase I, whereas neither was released by digestion with micrococcal nuclease, suggesting that the labeled molecules, like those of endogenous H6, were bound primarily to the core particles in transcriptionally competent portions of the genome. 2) Salt extraction curves were similar for both the labeled and unlabeled proteins, although about 15% of the labeled molecules were bound to the chromatin more loosely than those of the endogenous H6. Taken together, these results suggest that chromatin contains specific, well defined sites to which H6 binds. Upon increasing the concentration of H6 in the incubation mixture, progressively greater numbers of low affinity, presumably nonspecific binding sites become occupied. This observation has important implications for studies in which nucleases are employed to probe chromatin structure, since they suggest that H6 molecules released from specific, high affinity sites by the action of the nuclease might rebind more loosely to other regions of the chromatin.

Binding of HMG-T to trout testis chromatin

When 125I-labeled HMG-T was incubated with trout testis nuclei under conditions of pH and ionic strength approximating those in vivo , most of the radioactivity bound to the chromatin. Most labeled non-nuclear proteins which were tested did not bind. Four large cyanogen bromide fragments of HMG-T each bound, suggesting that HMG-T interacts with chromatin along most of its length. Trout testis chromatin contains two populations of HMG-T molecules which differ in their extractability with NaCl solutions; the 125I-labeled protein equilibrated mainly with the more readily extracted population. HMG-T also bound to nuclease-treated chromatin, an observation with important implications for studies in which nucleases are employed to probe chromatin structure.

Microinjection of the nonhistone chromosomal protein hmg1 into bovine fibroblasts and hela cells

The nonhistone chromosomal protein HMG1 associated rapidly with the nuclei of HeLa cells and bovine fibroblasts following its introduction into the cytoplasm by red cell-mediated microinjection. A number of non-nuclear proteins, on the other hand, failed to concentrate in HeLa or bovine fibroblast nuclei. Autoradiography of thin sections showed that 1251-labeled HMG1 localized within nuclei, and further established that it remained associated with metaphase chromosomes at mitosis. When uninjected HeLa cells were fused with 125l-HMG1-injected HeLa cells, the labeled molecules equilibrated between nuclei within 12 hr. Similar results were obtained with bovine fibroblasts, indicating that a dynamic equilibrium exists between HMG1 and chromatin within living cells. Electrophoresis of 125l-HMG1 retrieved from HeLa cells or bovine fibroblasts up to 48 hr after injection showed that more than 80% of the molecules were intact. Autoradiographic analysis of cells fixed over a period of several days after injection produced apparent half-lives for 125l-HMG1 of 80 hr in HeLa cells and 100 hr in bovine fibroblasts.