Cleavage/polyadenylation factor IA associates with the carboxyl-terminal domain of RNA polymerase II in Saccharomyces cerevisiae.

The carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II plays an important role in transcription and processing of the nascent transcript by interacting with both transcription and RNA processing factors. We show here that the cleavage/polyadenylation factor IA of Saccharomyces cerevisiae directly contacts CTD. First by affinity chromatography experiments with yeast extracts we demonstrate that the Rna15p, Rna14p, and Pcf11p subunits of this complex are associated with phosphorylated CTD. This interaction is confirmed for Rna15p by yeast two-hybrid analysis. Second, Pcf11p, but not Rna15p, is shown to directly contact phosphorylated CTD based on in vitro binding studies with recombinant proteins. These findings establish a direct interaction of cleavage/polyadenylation factor IA with the CTD. Furthermore, a quantitative analysis of transcription run-on performed on temperature-sensitive mutant strains reveals that the lack of either functional Rna14p or Pcf11p affects transcription termination more severely than the absence of a functional Rna15p. Moreover, these data reinforce the concept that CTD phosphorylation acts as a regulatory mechanism in the maturation of the primary transcript.

A clinically variant fibrosis syndrome in a Turkish family maps to the CFEOM1 locus on chromosome 12.

OBJECTIVES: To describe the phenotype of a Turkish family with variably expressed congenital fibrosis of the extraocular muscles (CFEOM), and to determine the genetic location of their disorder. METHODS: Participants were examined and had blood extracted for genetic analysis. The clinical features of the family's disorder were studied, and the disorder was tested for linkage to the 3 known CFEOM loci (CFEOM1, CFEOM2, and CFEOM3). RESULTS: Twenty-nine affected and 31 unaffected family members participated in the study. Eighteen affected individuals had congenital bilateral ptosis and restrictive infraductive (downward) ophthalmoplegia, consistent with the published descriptions of classic CFEOM families linked to the CFEOM1 locus. Eleven affected individuals, however, had eye(s) in a neutral primary position, residual upgaze, and/or absence of ptosis, thus deviating from previous descriptions of CFEOM1-linked families. Analysis of the autosomal dominant variably expressed disorder in this family revealed linkage to the CFEOM1 locus on chromosome 12 with a maximum lod score of 10.8 at D12S85. CONCLUSIONS: This Turkish family segregates a variably expressed form of CFEOM that most closely resembles CFEOM3-linked CFEOM, but maps to the CFEOM1 locus. CLINICAL RELEVANCE: These data establish that there is much greater phenotypic heterogeneity at the CFEOM1 locus than previously reported, and this may blur our ability to distinguish the different CFEOM loci based solely on clinical presentation. Arch Ophthalmol. 2000;118:1090-1097

Evidence of genetic heterogeneity in autosomal recessive congenital fibrosis of the extraocular muscles.

PURPOSE: Autosomal recessive congenital fibrosis of the extraocular muscles (CFEOM2) has been described in families from Saudi Arabia. Affected individuals have ptosis and exotropic ophthalmoplegia, and their disease has been mapped to chromosome 11q13. Here, we describe the phenotypic findings in a similarly affected Yemenite family and analyze the family for linkage to the CFEOM2 locus, as well as to the autosomal dominant CFEOM1 and CFEOM3 loci on chromosomes 12cen and 16q24, respectively. METHODS: The family was ascertained through two affected daughters. There are four unaffected siblings, and the parents are consanguineous. Each family member was examined, and linkage analysis was performed using markers from the CFEOM1, CFEOM2, and CFEOM3 loci. RESULTS: Both affected daughters have congenital bilateral ophthalmoplegia. The 15-month-old proband has restrictive exotropia. She fixates with either eye in abduction and with a compensatory head turn to the opposite side. Her 4-year-old sister has a small exotropia and severely limited eye movements. All other family members have normal ophthalmologic examinations. Genetic analysis excluded linkage of the family's disease to the CFEOM2 and CFEOM3 loci. A lod score of 2.0 (the maximum possible, given the family size and structure), was obtained at the CFEOM1 locus, and the alleles reduced to homozygosity in both affected daughters and none of the other children. CONCLUSIONS: These data establish that there is genetic heterogeneity in autosomal recessive CFEOM and suggest that this second recessive locus may be allelic to the autosomal dominant CFEOM1 locus at 12cen.

Stepwise in vitro affinity maturation of Vitaxin, an alphav beta3-specific humanized mAb.

A protein engineering strategy based on efficient and focused mutagenesis implemented by codon-based mutagenesis was developed. Vitaxin, a humanized version of the antiangiogenic antibody LM609 directed against a conformational epitope of the alphav beta3 integrin complex, was used as a model system. Specifically, focused mutagenesis was used in a stepwise fashion to rapidly improve the affinity of the antigen binding fragment by greater than 90-fold. In the complete absence of structural information about the Vitaxin-alphav beta3 interaction, phage-expressed antibody libraries for all six Ig heavy and light chain complementarity-determining regions were expressed and screened by a quantitative assay to identify variants with improved binding to alphav beta3. The Vitaxin variants in these libraries each contained a single mutation, and all 20 amino acids were introduced at each complementarity-determining region residue, resulting in the expression of 2,336 unique clones. Multiple clones displaying 2- to 13-fold improved affinity were identified. Subsequent expression and screening of a library of 256 combinatorial variants of the optimal mutations identified from the primary libraries resulted in the identification of multiple clones displaying greater than 50-fold enhanced affinity. These variants inhibited ligand binding to receptor more potently as demonstrated by inhibition of cell adhesion and ligand competition assays. Because of the limited mutagenesis and combinatorial approach, Vitaxin variants with enhanced affinity were identified rapidly and required the synthesis of only 2,592 unique variants. The use of such small focused libraries obviates the need for phage affinity selection approaches typically used, permitting the use of functional assays and the engineering of proteins expressed in mammalian cell culture.

Coupling termination of transcription to messenger RNA maturation in yeast.

The direct association between messenger RNA (mRNA) 3'-end processing and the termination of transcription was established for the CYC1 gene of Saccharomyces cerevisiae. The mutation of factors involved in the initial cleavage of the primary transcript at the poly(A) site (RNA14, RNA15, and PCF11) disrupted transcription termination at the 3' end of the CYC1 gene. In contrast, the mutation of factors involved in the subsequent polyadenylation step (PAP1, FIP1, and YTH1) had little effect. Thus, cleavage factors link transcription termination of RNA polymerase II with pre-mRNA 3'-end processing.

Heterolocalism: an alternative model of the sociospatial behaviour of immigrant ethnic communities.

"This paper evaluates critically the applicability of the well-known assimilation and pluralist models to the contemporary ethnic landscape of the U.S.... We then consider an alternative model, labelled heterolocalism, which suggests that members of certain newly arrived groups may be able to sustain their identity as an ethnic community despite immediate or rapid spatial dispersion. The applicability of the heterolocal perspective to non-metropolitan and transnational phenomena is evaluated in subsequent sections of the paper."

Transcriptional termination signals for RNA polymerase II in fission yeast.

Transcription 'run-on' (TRO) analysis using permeabilized yeast cells indicates that transcription terminates between 180 and 380 bp downstream of the poly(A) site of the Schizosaccharomyces pombe ura4 gene. Two signals direct RNA polymerase II (pol II) to stop transcription: the previously identified 3' end formation signals located close to the poly(A) site and an additional downstream element (DSE) located at the region of termination. The downstream signal (135 bp) appears to act by pausing the elongating polymerase. TRO analysis indicates that elevated levels of transcribing polymerases accumulate over the DSE and that removal of this signal leads to transcription proceeding beyond the normal termination region. Furthermore, when inserted between two competing polyadenylation signals, this DSE increases the utilization of upstream poly(A) sites in vivo. We show that polymerase pausing over an extended region of template ensures termination of pol II transcription close to the poly(A) site.

Identification of duck T lymphocytes using an anti-human T cell (CD3) antiserum.

Duck lymphocytes have not been classified into cells resembling B or T cells of mammals. Reagents used in the past to identify lymphocyte populations in other species have not been useful for this purpose and antibodies raised to duck immunoglobulin bind in high proportions to blood and organ lymphocytes of ducks as well as to their red blood cells. Here we report that a polyclonal rabbit antiserum reacting to the CD3 marker on human T cells has been used to identify duck T lymphocytes. These antibodies react with the intracytoplasmic portion of the human CD3 epsilon chain (amino acids 156-168), an epitope highly conserved between mammals. Immunohistochemical staining with this antiserum of sections of duck lymphoid organs and FACScan analysis of duck lymphoid cell suspensions identified a population of duck lymphocytes with a staining pattern similar to that seen for mammalian T cells. This anti-human CD3 immunoprecipitated a 23 kDa protein from a duck lymphoblast lysate: a size similar to the human CD3 epsilon chain. This is the first direct identification of duck T lymphocytes.

Activation domains of transcription factors mediate replication dependent transcription from a minimal HIV-1 promoter.

Transcription from a minimal HIV-1 promoter containing the three Sp1 binding sites and TATA box can be activated without Tat by template DNA replication. Here we show that this activation can also be mediated by recombinant GAL4 fusion proteins containing the activation domains of Sp1, VP16 or CTF (or by full-length GAL4) targeted to the HIV-1 promoter by replacing the Sp1 sites with five GAL4 binding sites. Thus Sp1 is not unique in its ability to mediate replication activated transcription, although the degree of processivity elicited by the different activators varied significantly from strongly processive (GAL4-VP16) to relatively non-processive (GAL4-Sp1 or -CTF). Processive GAL4-VP16-activated transcription, but not efficient initiation, required multiple GAL4 binding sites. In the presence of Tat, transcription with GAL4-SP1 and GAL4-CTF was further activated (principally at the level of processivity) but GAL4-VP16-potentiated transcription was only slightly stimulated. The Tat-dependent switch from non-processive to fully processive transcription was particularly marked for GAL4-Sp1, an effect which may be relevant to the selection of Sp1 binding sites by the HIV-1 promoter.

The v-sis oncoprotein loses transforming activity when targeted to the early Golgi complex.

The location of autocrine interactions between the v-sis protein and PDGF receptors remains uncertain and controversial. To examine whether receptor-ligand interactions can occur intracellularly, we have constructed fusion proteins that anchor v-sis to specific intracellular membranes. Fusion of a cis-Golgi retention signal from a coronavirus E1 glycoprotein to v-sis protein completely abolished its transforming ability when transfected into NIH3T3 cells. Fusion proteins incorporating mutations in this retention signal were not retained within the Golgi complex but instead were transported to the cell surface, resulting in efficient transformation. All chimeric proteins were shown to dimerize properly. Derivatives of some of these constructs were also constructed bearing the cytoplasmic tail from the glycoprotein of vesicular stomatitis virus (VSV-G). These constructs allowed examination of subcellular localization by double-label immunofluorescence, using antibodies that distinguish between the extracellular PDGF-related domain and the VSV-G cytoplasmic tail. Colocalization of sis-E1-G with Golgi markers confirmed its targeting to the early Golgi complex. The sis-E1 constructs, targeted to the early Golgi complex, exhibited no proteolytic processing whereas the mutant forms of sis-E1 exhibited normal proteolytic processing. Treatment with suramin, a polyanionic compound that disrupts ligand/receptor interactions at the cell surface, was able to revert the transformed phenotype induced by the mutant sis-E1 constructs described here. Our results demonstrate that autocrine interactions between the v-sis oncoprotein and PDGF receptors within the early Golgi complex do not result in functional signal transduction. Another v-sis fusion protein was constructed by attaching the transmembrane domain and COOH-terminus of TGN38, a protein that localizes to the trans-Golgi network (TGN). This construct was primarily retained intracellularly, although some of the fusion protein reached the surface. Deletion of the COOH-terminal region of the TGN38 retention signal abrogated the TGN-localization, as evidenced by very prominent cell surface localization, and resulted in increased transforming activity. The behavior of the sis-TGN38 derivatives is discussed within the context of the properties of TGN38 itself, which is known to recycle from the cell surface to the TGN.

Neighborhood context and residential mobility.

This paper extends the search for neighborhood contextual effects to residential mobility. We propose that neighborhood consists of subjective and objective domains, both of which are crosscut by substantive (social/physical) and temporal (current/change) dimensions. Measures of neighborhood characteristics consistent with our conceptualization are used to estimate the impact of context on mobility thoughts and on actual mobility in a sample of Nashville residents. Although individual statuses such as age and tenure remain important antecedents of mobility, subjective features of neighborhood context also play a role--albeit limited and indirect--in the decision to move or to stay.

The v-sis protein retains biological activity as a type II membrane protein when anchored by various signal-anchor domains, including the hydrophobic domain of the bovine papilloma virus E5 oncoprotein.

Membrane-anchored forms of the v-sis oncoprotein have been previously described which are oriented as type I transmembrane proteins and which efficiently induce autocrine transformation. Several examples of naturally occurring membrane-anchored growth factors have been identified, but all exhibit a type I orientation. In this work, we wished to construct and characterize membrane-anchored growth factors with a type II orientation. These experiments were designed to determine whether type II membrane-anchored growth factors would in fact exhibit biological activity. Additionally, we wished to determine whether the hydrophobic domain of the E5 oncoprotein of bovine papilloma virus (BPV) can function as a signal-anchor domain to direct type II membrane insertion. Type II derivatives of the v-sis oncoprotein were constructed, with the NH2 terminus intracellular and the COOH terminus extracellular, by substituting the NH2 terminal signal sequence with the signal-anchor domain of a known type II membrane protein. The signal-anchor domains of neuraminidase (NA), asialoglycoprotein receptor (ASGPR) and transferrin receptor (TR) all yielded biologically active type II derivatives of the v-sis oncoprotein. Although transforming all of the type II signal/anchor-sis proteins exhibited a very short half-life. The short half-life exhibited by the signal/anchor-sis constructs suggests that, in some cases, cellular transformation may result from the synthesis of growth factors so labile that they activate undetectable autocrine loops. The E5 oncoprotein encoded by BPV exhibits amino acid sequence similarity with PDGF, activates the PDGF beta-receptor, and thus resembles a miniature membrane-anchored growth factor with a putative type II orientation. The hydrophobic domain of the E5 oncoprotein, when substituted in place of the signal sequence of v-sis, was indistinguishable compared with the signal-anchor domains of NA, TR, and ASGPR, demonstrating its ability to function as a signal-anchor domain. NIH 3T3 cells transformed by the signal/anchor-sis constructs exhibited morphological reversion upon treatment with suramin, indicating a requirement for ligand/receptor interactions in a suramin-sensitive compartment, most likely the cell surface. In contrast, NIH 3T3 cells transformed by the E5 oncoprotein did not exhibit morphological reversion in response to suramin.

Intracellular retention of membrane-anchored v-sis protein abrogates autocrine signal transduction.

An important question regarding autocrine transformation by v-sis is whether intracellularly activated PDGF receptors are sufficient to transform cells or whether activated receptor-ligand complexes are required at the cell surface. We have addressed this question by inhibiting cell surface transport of a membrane-anchored v-sis protein utilizing the ER retention signal of the adenoviral transmembrane protein E3/19K. A v-sis fusion protein containing this signal was retained within the cell and not transported to the cell surface as confirmed by immunofluorescent localization experiments. Also, proteolytic maturation of this protein was suppressed, indicating inefficient transport to post-Golgi compartments of the secretory pathway. When compared with v-sis proteins lacking a functional retention signal, the ER-retained protein showed a diminished ability to transform NIH 3T3 cells, as measured by the number and size of foci formed. In newly established cell lines, the ER-retained protein did not down-regulate PDGF receptors. However, continued passage of these cells selected for a fully transformed phenotype exhibiting downregulated PDGF receptors and proteolytically processed v-sis protein. These results indicate that productive autocrine interactions occur in a post-ER compartment of the secretory pathway. Transport of v-sis protein beyond the Golgi correlated with acquisition of the transformed phenotype. Furthermore, suramin treatment reversed transformation and upregulated the expression of cell surface PDGF receptors, suggesting an important role for receptor-ligand complexes localized to the cell surface.

Multiple SP1 binding sites confer enhancer-independent, replication-activated transcription of HIV-1 and globin gene promoters.

We demonstrate that multiple SP1 protein:DNA binding sites confer enhancer-independent activation on the HIV-1 and globin gene promoters. This activation process can be achieved either by DNA replication of the promoter-containing plasmid or by high concentrations of input plasmid DNA used in the transfections. In the case of HIV-1, the three SP1 sites adjacent to the promoters TATA box are essential for this activation process. Furthermore, the human beta globin gene, which is normally dependent on a linked enhancer for transcriptional activity, can be made enhancer independent by insertion of SP1 binding sites adjacent to its TATA box. We speculate that (SP1)n-TATA type RNA polymerase II promoters may be generally permissive when present on actively replicating DNA templates and that this property of the HIV-1 promoter may be of importance to the activation of the DNA provirus in latently infected T cells.

Membrane-anchored form of v-sis/PDGF-B induces mitogenesis without detectable PDGF receptor autophosphorylation.

The v-sis protein is structurally and functionally related to PDGF. Forms of the v-sis protein which are anchored to the cell membrane via the transmembrane domain of the vesicular stomatitis virus G protein have been previously described (Hannink, M., and D.J. Donoghue. 1986. J. Cell Biol. 103:2311-2322). Several of these fusion proteins were shown to interact productively with the PDGF receptor (PDGFR) based on their ability to transform NIH 3T3 cells. In this report, we further characterized one of these membrane-anchored v-sis proteins, designated v-sis239-G. The gene encoding v-sis239-G was placed under control of the Drosophila melanogaster hsp70 promotor and synthesis of this protein was shown to induce a mitogenic response in NIH 3T3 cells. Unexpectedly, v-sis239-G did not induce detectable autophosphorylation of the PDGFR, in contrast to a similarly expressed secreted form of the v-sis protein. Thus, it appears that a PDGFR-mediated mitogenic response may be dissociated from detectable receptor autophosphorylation. Furthermore, induced synthesis of v-sis239-G was shown to lead to c-fos expression even in the absence of detectable receptor autophosphorylation. Interestingly, a nonmitogenic membrane-anchored form of the v-sis protein, designated v-sis239-G338, also induced c-fos without receptor autophosphorylation. These results raise interesting questions regarding the roles of autophosphorylation and c-fos induction in PDGFR-mediated signal transduction and suggest the possibility of an autophosphorylation-independent signal transduction pathway.

Is neighborhood racial succession place-specific?

This paper evaluates the geographic generality of the succession model of neighborhood racial change for the period 1970 to 1980. Using census data on racially mixed tracts, we determine whether white-to-black compositional shifts were equally common across the four regions and 58 central cities in our sample. Substantial variation occurred by region in the incidence and magnitude of racial succession; tracts in western cities departed most markedly from expectations. Even in other regions, some cities experienced more numerous instances of stability and displacement than of succession. These region and city effects persist when neighborhood characteristics believed to influence racial transition are controlled.

Xenopus homolog of the mos protooncogene transforms mammalian fibroblasts and induces maturation of Xenopus oocytes.

The oncogene v-mos transforms mammalian fibroblasts and encodes a serine/threonine protein kinase. Expression of the c-mos protooncogene is most abundant in germ cells, suggesting a normal role for c-mos in meiosis. Here we describe the isolation of cDNA clones containing the complete coding region of the Xenopus laevis homolog of c-mos (mosxe). The mosxe gene is transforming when introduced into murine NIH 3T3 cells, and transformation is abrogated by a lysine-to-arginine mutation in the canonical ATP-binding site. Microinjection of in vitro transcribed mosxe RNA into prophase-arrested Xenopus oocytes causes a resumption of meiosis, leading to germinal vesicle breakdown and oocyte maturation. Oocyte maturation was not observed after microinjection of in vitro transcribed mosxe RNA encoding the lysine-to-arginine mutation. These results demonstrate that the mosxe-encoded protein can induce progression through the cell cycle for both meiotic and mitotic cells and that this property is dependent on the presumptive ATP-binding domain in the protein kinase.

Stability and change in an urban homeless population.

Few studies have produced the over-time observational data needed to draw valid conclusions about changes in urban homeless populations during the 1980s. One place for which such data exist is Nashville, Tennessee. An ongoing series of enumerations lends little support to Nashvillians' perception that the number of homeless in their city is growing rapidly. Enumeration results also (1) contradict expectations regarding the rise of "new homeless" groups and (2) show two types of spatial redistribution--from indoor to outdoor and core to peripheral locations--to be under way. The applicability of the enumeration methodology to other communities is discussed, as are the discrepancies between purported and measured demographic changes in homelessness.

The alternatively spliced exon of the platelet-derived growth factor A chain encodes a nuclear targeting signal.

We have previously shown that the SIS/platelet-derived growth factor B chain contains a nuclear targeting signal near its C terminus. Here we show that the platelet-derived growth factor A chain also contains a nuclear targeting signal encoded by an exon which is subject to alternative splicing. This sequence is capable of targeting a nonsecreted form of the A chain to the nucleus and can also target the cytoplasmic proteins dihydrofolate reductase, chloramphenicol acetyltransferase, and pyruvate kinase to the nucleus.