A variety of proteins involved in gene expression have been localized within mammalian cell nuclei in a speckled distribution that predominantly corresponds to interchromatin granule clusters (IGCs). We have applied a mass spectrometry strategy to identify the protein composition of this nuclear organelle purified from mouse liver nuclei. Using this approach, we have identified 146 proteins, many of which had already been shown to be localized to IGCs, or their functions are common to other already identified IGC proteins. In addition, we identified 32 proteins for which only sequence information is available and thus these represent novel IGC protein candidates. We find that 54% of the identified IGC proteins have known functions in pre-mRNA splicing. In combination with proteins involved in other steps of pre-mRNA processing, 81% of the identified IGC proteins are associated with RNA metabolism. In addition, proteins involved in transcription, as well as several other cellular functions, have been identified in the IGC fraction. However, the predominance of pre-mRNA processing factors supports the proposed role of IGCs as assembly, modification, and/or storage sites for proteins involved in pre-mRNA processing.

Septins are a family of conserved proteins implicated in a variety of cellular functions such as cytokinesis and vesicle trafficking, but their properties and modes of action are largely unknown. Here we now report findings of immunocytochemical and biochemical characterization of a mammalian septin, MSF-A. Using an antibody specific for MSF subfamily proteins, MSF-A was found to be expressed predominantly in mammary human mammary epithelial cells (HMEC). MSF-A was associated with microtubules in interphase HMEC cells as it localized with the mitotic spindle and the bundle of microtubule at midzone during mitosis. Biochemical analysis revealed direct binding of MSF-A with polymerized tubulin through its central region containing guanine nucleotide-interactive motifs. GTPase activity, however, was not required for the association. Conditions that disrupt the microtubule network also disrupted the MSF-A-containing filament structure, resulting in a punctate cytoplasmic pattern. Depletion of MSF-A using small interfering RNAs caused incomplete cell division and resulted in the accumulation of binucleated cells. Unlike Nedd5, an MSF mutant deficient in GTPase activity forms filament indistinguishable from that of the wild type in COS cells. These results strongly suggest that septin filaments may interact not only with actin filaments but also with microtubule networks and that GTPase activity of MSF-A is not indispensable to incorporation of MSF-A into septin filaments.Septins, a family of heteropolymeric filament-forming proteins, were originally discovered in yeast to be essential for budding, and have since been identified in most eukaryotic organisms, with the exception of plants (for review, see Refs.

Abstract. Here, we describe the cloning and characterization of ScII, the second most abundant protein after topoisomerase II, of the chromosome scaffold fraction to be identified. ScII is structurally related to a protein, Smclp, previously found to be required for accurate chromosome segregation in Saccharomyces cerevisiae. SclI and the other members of the emerging family of SMCl-like proteins are likely to be novel ATPases, with NTP-binding A and B sites separated by two lengthy regions predicted to form an a-helical coiled-coil. Analysis of the SclI B site predicted that SclI might use ATP by a mechanism similar to the bacterial recN DNA repair and recombination enzyme. SclI is a mitosis-specific scaffold protein that colocalizes with topoisomerase II in mitotic chromosomes. However, SclI appears not to be associated with the interphase nuclear matrix. ScII might thus play a role in mitotic processes such as chromosome condensation or sister chromatid disjunction, both of which have been previously shown to involve topoisomerase II.

We show that the 3¢ boundary of the chicken b-globin locus bears striking structural similarities to the 5¢ boundary. In erythroid cells a clear transition in DNase I sensitivity of chromatin at the 3¢ end of the locus is observed, the location of this transition is marked by a constitutive DNase I hypersensitive site (HS), and DNA spanning this site has the enhancerblocking capacity of an insulator. This HS contains a binding site for the transcription factor CTCF. As in the case of the 5¢ insulator, the CTCF site is both necessary and suf®cient for the enhancer-blocking activity of the 3¢ boundary. The position of this insulator is consistent with our proposal that it may function to maintain the distinct regulatory programs of the globin genes and their closely appended 3¢ neighbor, an odorant receptor gene. We conclude that both boundaries of the chicken b-globin domain are capable of playing functionally similar roles and that the same protein is a necessary component of the molecular mechanism through which these boundaries are de®ned.

Genetics. In the article "Conservation of sequence and structure flanking the mouse and human ␤-globin loci: The ␤-globin genes are embedded within an array of odorant receptor genes" by Michael Bulger, J. Hikke von Doorninck, Noriko Saitoh, Agnes Telling, Catherine Farrell, M. A. Bender, Gary Felsenfeld, Richard Axel, and Mark Groudine, which appeared in number 9, April 27, 1999 of Proc. Natl. Acad. Sci. USA (96,(5129)(5130)(5131)(5132)(5133)(5134), the authors request that the following change be noted. Due to a printer's error, the author's name, J. Hikke von Doorninck, is misspelled and should be J. Hikke van Doorninck.Neurobiology. In the article "The effect of dynamic synapses on spatiotemporal receptive fields in visual cortex" by Ö mer B. Artun, Harel Z. Shouval, and Leon N. Cooper, which appeared in number 20, September 29, 1998, of Proc. Natl. Acad. Sci. USA (95, 11999 -12003), the legend for Neurobiology. In the article "Fluctuations and stimulusinduced changes in blood flow observed in individual capillaries in layers 2 through 4 of rat neocortex" by David Kleinfeld, Partha P. Mitra, Fritjof Helmchen, and Winfried Denk, which appeared in number 26, December 22, 1998, of Proc. Natl. Acad. Sci. USA, the authors request that the following correction be noted. Two-photon laser scanning microscopy was used to measure the flow of red blood cells in capillaries in the living brain. In this manuscript, we also reported the length-constant for attenuation of the signal (i.e., two-photon excited fluorescence) as a function of penetration depth into the cortex. As a result of an error in our analysis, the length-constants for each data set were overestimated. The correct value for the data of Fig. 1 is 100 m (instead of 140 m), and the range of values over all data sets is from 100 m to 170 m (instead of 120 m to 220 m). We apologize for any inconvenience caused by our error. None of the other results or conclusions in the original manuscript are affected. Contributed by Gary Felsenfeld, February 24, 1999 ABSTRACT In mouse and human, the ␤-globin genes reside in a linear array that is associated with a positive regulatory element located 5 to the genes known as the locus control region (LCR). The sequences of the mouse and human ␤-globin LCRs are homologous, indicating conservation of an essential function in ␤-globin gene regulation. We have sequenced regions f lanking the ␤-globin locus in both mouse and human and found that homology associated with the LCR is more extensive than previously known, making up a conserved block of Ϸ40 kb. In addition, we have identified DNaseI-hypersensitive sites within the newly sequenced regions in both mouse and human, and these structural features also are conserved. Finally, we have found that both mouse and human ␤-globin loci are embedded within an array of odorant receptor genes that are expressed in olfactory epithelium, and we also identify an olfactory receptor gene located 3 of the ␤-globin locus in chicken. The data demonstrate an evolutionarily conserved genom...