Simple sequences are stretches of DNA which consist of only one, or a few tandemly repeated nucleotides, for example poly (dA) X poly (dT) or poly (dG-dT) X poly (dC-dA). These two types of simple sequence have been shown to be repetitive and interspersed in many eukaryotic genomes. Several other types have been found by sequencing eukaryotic DNA. In this report we have undertaken a systematical survey for simple sequences. We hybridized synthetical simple sequence DNA to genome blots of phylogenetically different organisms. We found that many, probably even all possible types of simple sequence are repetitive components of eukaryotic genomes. We propose therefore that they arise by common mechanisms namely slippage replication and unequal crossover and that they might have no general function with regards to gene expression. This latter inference is supported by the fact that we have detected simple sequences only in the metabolically inactive micronucleus of the protozoan Stylonychia, but not in the metabolically active macronucleus which is derived from the micronucleus by chromosome diminution.

No abstract

We have previously described a SWI/SNF-related protein complex (PYR complex) that is restricted to definitive (adult-type) hematopoietic cells and that specifically binds DNA sequences containing long stretches of pyrimidines. Deletion of an intergenic DNA-binding site for this complex from a human ␤-globin locus construct results in delayed human ␥-to ␤-globin switching in transgenic mice, suggesting that the PYR complex acts to facilitate the switch. We now show that PYR complex DNA-binding activity also copurifies with subunits of a second type of chromatin-remodeling complex, nucleosome-remodeling deacetylase (NuRD), that has been shown to have both nucleosome-remodeling and histone deacetylase activities. Gel supershift assays using antibodies to the ATPase-helicase subunit of the NuRD complex, Mi-2 (CHD4), confirm that Mi-2 is a component of the PYR complex. In addition, we show that the hematopoietic cell-restricted zinc finger protein Ikaros copurifies with PYR complex DNA-binding activity and that antibodies to Ikaros also supershift the complex. We also show that NuRD and SWI/SNF components coimmunopurify with each other as well as with Ikaros. Competition gel shift experiments using partially purified PYR complex and recombinant Ikaros protein indicate that Ikaros functions as a DNA-binding subunit of the PYR complex. Our results suggest that Ikaros targets two types of chromatin-remodeling factors-activators (SWI/SNF) and repressors (NuRD)-in a single complex (PYR complex) to the ␤-globin locus in adult erythroid cells. At the time of the switch from fetal to adult globin production, the PYR complex is assembled and may function to repress ␥-globin gene expression and facilitate ␥-to ␤-globin switching.

ABSTRACTnaked DNA, and that it is the cooperative binding of histone Hi that is responsible for the folding of the thin chromosome fiber. The results of four independent experimental approaches provide evidence that supports this view: (i) competition between long and short nucleosome chains for histone Hi analyzed by filter binding; (ii) the distribution of histone Hi in mixtures of long and short chromosome fibers separated by sucrose gradient velocity sedimentation; (iii) the sedimentation behavior of long chromosome fiber fragments as a function of NaCl concentration in the range of the transition; (iv) electron microscopy of chromosome fibers above and below the transition. MATERIALS AND METHODSCell nuclei were isolated from bovine lymphocytes (13) and stored in 2 mM MgCl2, 5 mM Tris.HCl (pH 7.5), and 66% (vol/vol) glycerol at -600 until needed, but not longer than 10 days. Radioactively labeled nuclei were obtained from bovine lymphocytes that had been stimulated by phytohemagglutinin P (Difco) in medium containing [3H]thymidine after 60 hr of incubation.Fragmentation of chromatin in nuclei by micrococcal nuclease (Boehringer or Worthington) was carried out at 00 in 0.2 M sucrose, 1 mM CaCl2, 5 mM Tris-HCI (pH 7.5), and either 80 or 60 mM NaCl at a concentration of 2.4-108 nuclei per ml. The digestion reaction was terminated and nuclei were lysed by gently adding the same volume of a solution containing 5 mM EDTA, 5 mM Tris-HCl (pH 7.5), and 80 or 60 mM NaCl. The nuclear debris was pelleted at 5000 X g for 8 min. The supernatant contained 50-80% of the nuclear DNA in the form of fragmented chromosome fibers. Histone-HI-depleted fiber fragments were made by the aid of tRNA (14). Histone HI was prepared by the trichloroacetic acid extraction procedure (15).The filter binding assay was carried out as described earlier (12). Stock solutions of histone H1 were diluted into 0.5 ml samples of 5 mM Tris-HCl (pH 7.5) and 40 mM NaCl containing equal weights of labeled and unlabeled histone-Hldepleted nucleosomes. After incubation for 30 min at 00, the reaction mixture was filtered through nitrocellulose membrane filters at a flow rate of 0. 1 ml-sec-'. The filters were washed three times with 0.7 ml of buffer, dried, and monitored for radioactivity. The values given are the means of three experiments; the standard deviations were less than 10% of the mean values. Filters retained 20 (+5)% of the histone-Hi-depleted nucleosome trimer (background).Sucrose gradient analyses were made by layering fragmented chromosome fiber samples (0.5 ml) on preformed linear gradients from 10 to 30% sucrose containing 1 mM sodium phosphate (pH 6.8), 0.2 mM EDTA, and NaCl at the same concentration as in the samples. Nitrocellulose tubes with 0.5 ml cushions of 86% (vol/vol) glycerol were spun in a Beckman SW 40 rotor at 3°. The gradients were analyzed with the use of a turbulence-free flow cell (ISCO).DNA sizes were analyzed electrophoretically on 1.4% agarose gels as previously described (16

Unlike conventional membrane proteins of the secretory pathway, proteins anchored to the cytoplasmic surface of membranes by hydrophobic sequences near their C termini follow a posttranslational, signal recognition particleindependent insertion pathway. Many such C-terminallyanchored proteins have restricted intracellular locations, but it is not known whether these proteins are targeted directly to the membranes in which they will ultimately reside. Here we have analyzed the intracellular sorting of the Golgi protein giantin, which consists of a rod-shaped 376-kDa cytoplasmic domain followed by a hydrophobic C-terminal anchor sequence. Unexpectedly, we find that giantin behaves like a conventional secretory protein in that it inserts into the endoplasmic reticulum (ER) and then is transported to the Golgi. A deletion mutant lacking a portion of the cytoplasmic domain adjacent to the membrane anchor still inserts into the ER but fails to reach the Golgi, even though this mutant has a stable folded structure. These findings suggest that the localization of a C-terminally-anchored Golgi protein involves at least three steps: insertion into the ER membrane, controlled incorporation into transport vesicles, and retention within the Golgi.