Chromatin In an attempt to understand the role of chromosomal proteins we have begun a study of erythroid differentiation using murine erythroleukemia cells transformed by Friend virus (FL cells) (5,6). FL cells offer a useful model system for the study of the role of chromosomal proteins because at least the terminal events of erythroid differentiation can be provoked in vitro. FL cells contain little or no hemoglobin or globin mRNA and do not display such characteristic erythroid antigens as glycophorin or spectrin. Addition of various compounds induces a terminal erythroid differentiation in these cells (7)(8)(9)(10)(11). This differentiation, which takes place over a period of 3-5 days, is characterized by the accumulation of globin mRNA and hemoglobin (12,13) and by the appearance of glycophorin and spectrin (ref. 14; H. Eisen, unpublished data). During the induction period the cells become smaller, globin RNA and protein synthesis is shut off, and, after two or three divisions, cell division ceases (15-17). Peterson and McConkey (18) compared the proteins of differentiating and nondifferentiating FL cells and observed some changes in the chromosomal proteins. We report here the preliminary characterization of a major chromatin protein that appears after induction of FL cells.MATERIALS AND METHODS Cells and Culture Methods. FL cells of clone F4N were obtained from W. Ostertag (19,20). The clone MA-1 was derived from the DBA/2 mastocytoma P815 obtained from P. Vassalli. The isolation and characterization of resistant cell lines F4+ and F4N+2 will be described elsewhere (H. Eisen and W. Ostertag, unpublished data). All cells were grown in suspension culture in minimal essential medium (21) Chromatin Purification. Chromatin was purified by a modification of the method of Bonner et al. (23). Cells (50 to 100 X 106) were harvested by centrifugation at 800 X g for 10 min, washed twice with phosphate-buffered saline, and disrupted by homogenization in hypotonic buffer (24) containing 1 mM phenylmethylsulfonylfluoride. Nuclei were sedimented at 1000 X g and washed once with the same buffer. The washed nuclei were lysed in 10 mM Tris-HCI (pH 8)/25 mM EDTA/ 0.5% Triton X-100/1 mM phenylmethylsulfonylfluoride and homogenized. After two washes with 5 mM Tris-HCI, pH 8, chromatin was sedimented through a solution of 65% sucrose in 5 mM Tris for 70 min. at 80,000 X g. The chromatin pellet was washed twice with 10 mM TrisMHCI (pH 7.4)/1 mM EDTA and resuspended in the same buffer either by sonication (three times for 15 sec in a MSE sonicator) or by a Sorvall omnimixer (90 sec at 25,000 rpm). Chromatin was quantitated on the basis of its DNA content by measuring its absorbance at 260 nm.Polyacrylamide Gel Electrophoresis. Chromatin proteins were analyzed on slab gels (25) by a modification of Laemmli's discontinuous buffer system (26,27). The running gel contained 15% acrylamide and 0.087% N',N-methylene bisacrylamide. The gels were stained in 0.1% Coomassie brilliant blue/10% acetic acid/50% methanol, destained...

The text of Table 1 contained several errors, which we regret. The correct version is printed below.

The protein IP25, which has previously been reported to accumulate in the chromatin during erythroid differentiation of Friend-virus-transformed erythroleukemia cells ( F L cells), is shown to behave like histone H1 without being structurally related to it. Like H1. IP25 is not released by digestion of F L cells nuclei with DNAse I. After micrococcal digestion IP25 and HI are differentially distributed in the nucleosome monomers and dimers. This distribution suggests an internucleosomal location for IP25 and H1. Different rates of digestion are observed between nuclei of differentiating and non-differentiating FL cells with both DNAse I and micrococcal nuclease. These differences could be due to the presence of IP25 in the chromatin of differentiating cells.I t is widely assumed that chromatin contains proteins involved in the regulation of gene expression. Before one can elaborate on the functions of such proteins it is necessary to know how they are inserted in the chromatin structure. Recent studies have led to a model of chromatin structure based on a repeating unit, termed nucleosome, which is composed of histones surrounded by 200 base pairs of D N A [l -31. The octamer contains two copies of the histones H2A, H2B. H 3 and H4. It is believed that HI does not contribute to the structure of the nucleosome but that it is bound to the DNA outside the units. The results of nuclease digestion analysis suggest that HI is associated with the region of DNA that links' adjacent nucleosomes [4-61. Thus H1 may be involved in the coiling or folding of nucleosome chains [7]. More information about chromatin structure in the neighbourhood of transcriptionally active genes has been obtained by examination of its sensitivity to nuclease. Several studies have shown that active genes are likely to be packaged by histones but that these histones are in an altered conformation which renders the associated D N A extremely sensitive to digestion by pancreatic DNAse I [8,9]. These findings provided a way of analyzing proteins which are associated with transcriptionally active genes.We have previously described a chromosomal protein. IP25, whose appearance is associated with the erythroid differentiation of Friend-virus-transformed erythroleukemia cells ( F L cells). It was shown thatIP25 is tightly bound to the chromatin and that it accumulates during the erythroid differentiation of the cells [lo]. The IP25 was shown to elute from the chromatin at NaCl concentrations greater than 0.5 M. In this respect it behaves like the H1 histones and not like the high-mobility-group proteins which are eluted from chromatin at 0.35 M NaCl 1111.We report here experiments designed to locate IP25 within the chromatin structure of FL cells. Our results indicate that IP25 is located on the transcriptionally inactive chromatin and that, like HI it is bound to internucleosomal regions. MATERIALS A N D METHODS Cells unu' Culture MtjtliodsFL cells from clone F4N were obtained from W. Ostertag [12]. The properties and selection of the resistant clone,...

The Escherichia coli chaperonin machine is composed of two members, GroEL and GroES. The GroEL chaperonin can bind 10-15% of E. coli's unfolded proteins in one of its central cavities and help them fold in cooperation with the GroES cochaperonin. Both proteins are absolutely essential for bacterial growth. Several large, lytic bacteriophages, such as T4 and RB49, use the host-encoded GroEL in conjunction with their own bacteriophageencoded cochaperonin for the correct assembly of their major capsid protein, suggesting a cochaperonin specificity for the in vivo folding of certain substrates. Here, we demonstrate that, when the cochaperonin of either bacteriophage T4 (Gp31) or RB49 (CocO) is expressed in E. coli, the otherwise essential groES gene can be deleted. Thus, it appears that, despite very little sequence identity with groES, the bacteriophageencoded Gp31 and CocO proteins are capable of replacing GroES in the folding of E. coli's essential, housekeeping proteins.

Escherichia coli mutants, called groNB, which block the growth of bacteriophage lambda at the level of action of the gene N product, have been isolated as survivors at 42 degrees C of bacteria carrying a) the defective prophage lambda bio11 i lambda cI857 delta H1 or b) the pcR1 plasmid containing the EcoRI immunity fragment of phage lambda cI857. In addition, groNB bacterial mutants have been isolated at 37 degrees C, as large colony formers in the presence of lambda i lambda cI h434, lambda i lambda cI h lambda, and lambda i lambda cI h80 phage. The groNB locus is located at 9 minute of the E. coli genetic map with the order of the neighboring loci being proC tsx groNB purE. Most groNB mutations isolated at 42 degrees C were found to interfere in addition with bacterial growth at low temperatures, since (a) the GroNB phenotypes of lambda growth inhibition and bacterial cold sensitivity cannot be separated by P1 transduction, and (b) some cold resistant revertants simultaneously become Gro+ for lambda growth. Lambda transducing phages carrying the groNB+ bacterial gene have been isolated. GroNB mutant bacteria lysogenized by the transducing phage acquire the Gro+ phenotype and simultaneously the cold resistant phenotype, suggesting that the groNB mutations are recessive to the wild-type gene.