Sequence variation and linkage disequilibrium in the human T-cell receptor beta (TCRB) locus.

The T-cell receptor (TCR) plays a central role in the immune system, and > 90% of human T cells present a receptor that consists of the alpha TCR subunit (TCRA) and the beta subunit (TCRB). Here we report an analysis of 63 variable genes (BV), spanning 553 kb of TCRB that yielded 279 single-nucleotide polymorphisms (SNPs). Samples were drawn from 10 individuals and represent four populations-African American, Chinese, Mexican, and Northern European. We found nine variants that produce nonfunctional BV segments, removing those genes from the TCRB genomic repertoire. There was significant heterogeneity among population samples in SNP frequency (including the BV-inactivating sites), indicating the need for multiple-population samples for adequate variant discovery. In addition, we observed considerable linkage disequilibrium (LD) (r(2) > 0.1) over distances of approximately 30 kb in TCRB, and, in general, the distribution of r(2) as a function of physical distance was in close agreement with neutral coalescent simulations. LD in TCRB showed considerable spatial variation across the locus, being concentrated in "blocks" of LD; however, coalescent simulations of the locus illustrated that the heterogeneity of LD we observed in TCRB did not differ markedly from that expected from neutral processes. Finally, examination of the extended genotypes for each subject demonstrated homozygous stretches of >100 kb in the locus of several individuals. These results provide the basis for optimization of locuswide SNP typing in TCRB for studies of genotype-phenotype association.

Characterization of differentially expressed genes in purified Drosophila follicle cells: toward a general strategy for cell type-specific developmental analysis.

Axis formation in Drosophila depends on correct patterning of the follicular epithelium and on signaling between the germ line and soma during oogenesis. We describe a method for identifying genes expressed in the follicle cells with potential roles in axis formation. Follicle cells are purified from whole ovaries by enzymatic digestion, filtration, and fluorescence-activated cell sorting (FACS). Two strategies are used to obtain complementary cell groups. In the first strategy, spatially restricted subpopulations are marked for FACS selection using a green fluorescent protein (GFP) reporter. In the second, cells are purified from animals mutant for the epidermal growth factor receptor ligand gurken (grk) and from their wild-type siblings. cDNA from these samples of spatially restricted or genetically mutant follicle cells is used in differential expression screens employing PCR-based differential display or hybridization to a cDNA microarray. Positives are confirmed by in situ hybridization to whole mounts. These methods are found to be capable of identifying both spatially restricted and grk-dependent transcripts. Results from our pilot screens include (i) the identification of a homologue of the immunophilin FKBP-12 with dorsal anterior expression in egg chambers, (ii) the discovery that the ecdysone-inducible nuclear hormone receptor gene E78 is regulated by grk during oogenesis and is required for proper dorsal appendage formation, and (iii) the identification of a Drosophila homologue of the human SET-binding factor gene SBF1 with elevated transcription in grk mutant egg chambers.

maelstrom is required for an early step in the establishment of Drosophila oocyte polarity: posterior localization of grk mRNA.

We describe a mutant, maelstrom, that disrupts a previously unobserved step in mRNA localization within the early oocyte, distinct from nurse-cell-to-oocyte RNA transport. Mutations in maelstrom disturb the localization of mRNAs for Gurken (a ligand for the Drosophila Egf receptor), Oskar and Bicoid at the posterior of the developing (stage 3-6) oocyte. maelstrom mutants display phenotypes detected in gurken loss-of-function mutants: posterior follicle cells with anterior cell fates, bicoid mRNA localization at both poles of the stage 8 oocyte and ventralization of the eggshell. These data are consistent with the suggestion that early posterior localization of gurken mRNA is essential for activation of the Egf receptor pathway in posterior follicle cells. Posterior localization of mRNA in stage 3-6 oocytes could therefore be one of the earliest known steps in the establishment of oocyte polarity. The maelstrom gene encodes a novel protein that has a punctate distribution in the cytoplasm of the nurse cells and the oocyte until the protein disappears in stage 7 of oogenesis.

Transposon insertions causing constitutive Sex-lethal activity in Drosophila melanogaster affect Sxl sex-specific transcript splicing.

Sex-lethal (Sxl) gene products induce female development in Drosophila melanogaster and suppress the transcriptional hyperactivation of X-linked genes responsible for male X-chromosome dosage compensation. Control of Sxl functioning by the dose of X-chromosomes normally ensures that the female-specific functions of this developmental switch gene are only expressed in diplo-X individuals. Although the immediate effect of X-chromosome dose is on Sxl transcription, during most of the life cycle "on" vs. "off" reflects alternative Sxl RNA splicing, with the female (productive) splicing mode maintained by a positive feedback activity of SXL protein on Sxl pre-mRNA splicing. "Male-lethal" (SxlM) gain-of-function alleles subvert Sxl control by X-chromosome dose, allowing female Sxl functions to be expressed independent of the positive regulators upstream of Sxl. As a consequence, SxlM haplo-X animals (chromosomal males) die because of improper dosage compensation, and SxlM chromosomal females survive the otherwise lethal effects of mutations in upstream positive regulators. Five independent spontaneous SxlM alleles were shown previously to be transposon insertions into what was subsequently found to be the region of regulated sex-specific Sxl RNA splicing. We show that these five alleles represent three different mutant types: SxlM1, SxlM3, and SxlM4. SxlM1 is an insertion of a roo element 674 bp downstream of the translation-terminating male-specific exon. SxlM3 is an insertion of a hobo transposon (not 297 as previously reported) into the 3' splice site of the male exon, and SxlM4 is an insertion of a novel transposon into the male-specific exon itself. We show that these three gain-of-function mutants differ considerably in their ability to bypass the sex determination signal, with SxlM4 being the strongest and SxlM1 the weakest. This difference is also reflected in effects of these mutations on sex-specific RNA splicing and on the rate of appearance of SXL protein in male embryos. Transcript analysis of double-mutant male-viable SxlM derivatives in which the SxlM insertion is cis to loss-of-function mutations, combined with other results reported here, indicates that the constitutive character of these SxlM alleles is a consequence of an alteration of the structure of the pre-mRNA that allows some level of female splicing to occur even in the absence of functional SXL protein. Surprisingly, however, most of the constitutive character of SxlM alleles appears to depend on the mutant alleles' responsiveness, perhaps greater than wild-type, to the autoregulatory splicing activity of the wild-type SXL proteins they produce.

Serum amyloid A changes high density lipoprotein's cellular affinity. A clue to serum amyloid A's principal function.

The affinity of high density lipoproteins (HDL), or HDL carrying serum amyloid A (HDL/SAA), for hepatocytes or peritoneal macrophages was examined, as part of an investigation exploring the principal function of SAA and how this may be related to amyloidogenesis. The binding results in conjunction with SAA's existence primarily on HDL during inflammation, and HDL's known "reverse cholesterol transport" function suggest a clear role for SAA in the afferent arm of the reverse cholesterol transport pathway during the process of inflammation. The presence of SAA reduced HDL's affinity for normal hepatocytes by a factor of 2. In contrast, HDL/SAA had a 3- to 4-fold higher affinity for macrophages than HDL alone. Furthermore, the number of binding sites for HDL/SAA increased on macrophages during inflammation, while decreasing on hepatocytes. The net effect was a significant shift in HDL cholesterol carrying capacity towards the macrophage. Competition experiments demonstrated that HDL/SAA is only half as effective as HDL in inhibiting radiolabeled HDL binding to macrophages. This is in keeping with the reduced apolipoprotein A-1 content in HDL/SAA. Strikingly, although HDL contains twice as much apolipoprotein A-1 as HDL/SAA, it is only one-tenth as effective as HDL/SAA in inhibiting radiolabeled HDL/SAA binding to macrophages. The latter results suggest that there is a specific SAA binding site on macrophages.

Effects of culture substrates and normal hepatic sinusoidal cells on in vitro hepatocyte synthesis of Apo-SAA.

Primary hepatocyte cultures synthesize apo-SAA upon stimulation with supernatant from lipopolysaccharide (LPS)-treated macrophages. The matrices on which the hepatocytes were grown influence their basal apo-SAA synthetic capability. Fibronectin was superior. Coculturing hepatocytes with hepatic sinusoidal cells did not adversely affect the ability of hepatocytes to synthesize and secrete apo-SAA into the culture medium. In 72 h, clear islands of endothelial cells nestled in layers of hepatocytes. Both apo-SAA and apo-SAA were made in considerable quantities but no evidence could be obtained that the apo-SAA were free of apo-A-1. The coculturing of hepatocytes with liver sinusoidal cells, the site of ultimate AA deposition, is a first step in establishing an in vitro system for AA amyloidogenesis.

48-Kilodalton intermediate-filament-associated protein in astrocytes.

We provide evidence that a protein of 48 kilodaltons (KD), recognized by a normal rabbit serum (F2N), is associated with intermediate filaments (IF) of astrocytes both in cell cultures and in situ. Immunofluorescence staining shows that the F2N serum gives a fibrous staining pattern similar to that seen with anti-serum to glial filament protein (GFP), a protein specific for IF of astrocytes, and that both proteins are present in the perinuclear fibrous aggregates of IF produced by treating the cells with colchicine. At the ultrastructural level the gold particles decorating the 48-KD protein are localized in clusters along the IF, whereas the gold particles decorating the GFP are localized on the IF in a linear pattern. This difference in distribution and the fact that the two proteins have different electrophoretic mobilities on SDS gels indicates that the 48-KD protein although associated with IF is different from GFP. The 48-KD protein appears to be a distinct, developmentally regulated intermediate-filament-associated protein (IFAP), different from other IFAPs reported to date and the first IFAP described in astrocytes. Its appearance in late developmental stages when motile astroblasts are changing into nonmotile stellate cells suggests that the 48-KD protein may be involved in cross-linking the GFP-containing IF to provide more tensile strength to the cytoplasm at the expense of flexibility.

Assembly of glial intermediate filament protein is initiated in the centriolar region.

Assembly of glial intermediate filament protein (GFP) into intermediate filaments (IF) was first detected by immunofluorescence in the perinuclear region of astrocytes differentiating in colony cultures before the rest of the cytoplasm was labeled. Double labeling with antisera specific for centrioles indicated that this site corresponds to the centriolar region. These studies suggest that the centriolar region plays an important role in the assembly of some types of IF as well as microtubules.

Are elevated serum amyloid A levels and amyloid-enhancing factor sufficient to induce inflammation-associated amyloid deposition?

During inflammation-associated amyloidosis two coincident factors, serum amyloid A (SAA) and amyloid-enhancing factor (AEF) are apparently necessary for amyloid A (AA) deposition. It is shown by passive transfer of cytokines, which stimulate SAA production, and AEF that these are not sufficient. A further factor(s) is necessary, which stems from the acute inflammatory response. Potential candidates are serum or tissue glycosaminoglycans.

Microtubule-organizing centers and cell migration: effect of inhibition of migration and microtubule disruption in endothelial cells.

We have previously shown that microtubule-organizing centers (MTOC's) become preferentially oriented towards the leading edge of migrating endothelial cells (EC's) at the margin of an experimentally induced wound made in a confluent EC monolayer. To learn more about the mechanism responsible for the reorientation of MTOC's and to determine whether a similar reorientation takes place when cell migration is inhibited, we incubated the wounded cultures with colcemid (C) and cytochalasin B (CB), which disrupt microtubules (MT's) and microfilaments (MF's), respectively. The results obtained showed that the MTOC reorientation can occur independent of cell migration since MTOC's reoriented preferentially toward the wound edge in the CB-treated cultures, even though forward migration of the EC was inhibited. In addition, the MTOC reorientation is inhibited by C, indicating that it requires an intact system of MT's and/or other intracellular structures whose distribution is dependent on that of MT's.

Astrocyte cell lineage. II. Mouse fibrous astrocytes and reactive astrocytes in cultures have vimentin- and GFP-containing intermediate filaments.

When cells from mouse neopallium are grown in colony cultures for 10-12 days, small cells with many processes, resembling normal fibrous astrocytes, form on top of the astrocyte precursor cells independently of the presence of dBcAMP in the culture medium. These cells are distinctly different from the much larger, previously described reactive astrocytes which also form in colony cultures and whose maturation is greatly enhanced by the presence of dBcAMP in the culture medium. Immunofluorescence studies showed that both vimentin-containing and glial filament protein (GFP)-containing intermediate filaments (IF) are present in the small normal fibrous astrocytes as well as in the larger reactive astrocytes. The vimentin-containing IF are assembled first in astrocyte precursor cells, whereas GFP-containing IF are assembled later toward the final stages of astrocyte differentiation both in vivo and in vitro. Thus in respect to the expression of the two types of IF, astrocyte differentiation in vitro closely resembles that in vivo. Parallel studies by electron microscopy showed that the vimentin-positive but GFP-negative astrocyte precursor cells contain single IF or small groups of IF, whereas in the more differentiated normal fibrous astrocytes and reactive astrocytes which are also GFP-positive, additional IF arranged in large bundles are present.

Distribution of microtubule organizing centers in migrating sheets of endothelial cells.

This study was designed to investigate the relationship between the position of the microtubule organizing center (MTOC) and the direction of migration of a sheet of endothelial cells (EC). Using immunofluorescence and phase microscopy the MTOC's of migrating EC were visualized as the cells moved into an in vitro experimental wound produced by mechanical denudation of part of a confluent monolayer culture. Although the MTOC's in nonmigrating EC were randomly positioned in relation to the nucleus, in migrating cells the position of the MTOC's changed so that 80% of the cells had the MTOC positioned in front of the nucleus toward the direction of movement of the endothelial sheet. This repositioning of the MTOC occurred within the first 4 h after wounding and was associated with the beginning of migration of EC's into the wounded area as seen by time-lapse cinemicrophotography. These studies focus attention on the MTOC as a cytoskeletal structure that may play a role in determining the direction of cell movement.

The reorganization of cytoskeletal fibre systems in spreading porcine endothelial cells in cultures.

Porcine aortic endothelial cells spreading on a glass substrate undergo characteristic changes in shape which can be classified into four distinct stages. To study the role of the cytoskeleton in cell spreading, we have examined the distribution of microtubules (MT), microfilaments (MF), and intermediate filaments (IF) at each of these stages by using immunofluorescence and antisera specific for tubulin, tropomyosin, myosin, and vimentin. The small round Stage I cells showed diffuse staining with four antisera. In the more flattened spreading Stage II cells, MT and IF were first observed in the perinuclear region while fibres straining positively for tropomyosin and myosin were first seen along the cell margin. Later the MT began to radiate out in all directions from the perinuclear region while the IF became localized in a region on one side of the nucleus. In the very flat Stage III cells with a circular outline, additional MT could be seen along and parallel to cell margin while the IF emanating from the perinuclear region and the tropomyosin and myosin positive fibres became concentrically distributed around the nucleus. In the very flat asymmetric Stage IV cells, both the MT and IF radiated out from the perinuclear region towards the cell periphery while most of the tropomyosin and myosin-positive fibres became reorganized so that they ran parallel to the edges of the cell. In addition several loci from which a number of the tropomyosin and myosin-containing fibres radiated also appeared at this stage. These results indicate that during spreading each of the three major fibre systems undergo extensive and specific reorganization which is well coordinated with changes in cell shape.

Localization of the neurofilament protein in neuroblastoma cells by immunofluorescent staining.

Neurofilament protein (54,000-56,000 daltons) has been localized in murine neuroblastoma cells by indirect immunofluorescent staining with antisera to purified calf brain neurofilament protein. In some cells with only short processes, specific staining of fibrous material was present in the perinuclear region while in other cells similar fibers, coiled to varying degrees, were present in other regions of the cytoplasm. In cells with longer processes a stained fiber extended throughout each process. The staining pattern observed followed the distribution of bundles of 100 A filaments as determined by electron microscopy. The fibers did not stain with antisera to tubulin or tropomyosin. The observations reported strongly indicate (i) that neurofilament protein isolated from calf brain is antigenically related to a component of the bundles of 100 A filaments in neuroblastoma cells, and (ii) that the neurofilament protein is an integral part of bundles of 100 A filaments in neuroblastoma cells, while neither tubulin nor tropomyosin is present in these bundles.

Antisera against electrophoretically purified tubulin stimulate colchicine-binding activity.

Several rabbit antisera have been prepared against reduced and alkylated, electrophoretically purified tubulin isolated from chick brain. These antisera give a single precipitin line in Ouchterlony double diffusion plates when tested against partially purified tubulin, and label specifically microtubule- and tubulin-containing structures, such as mitotic spindles, cilia, and vinblastine-induced crystals, in a variety of cells. The same antisera also display the unique ability to stimulate the colchicine-binding activity of tubulin preparations from chick brain and Chinese hamster ovary tissue culture cells. This specific stimulation of colchicine binding activity is also obtained with the gamma globulin fractions purified by ammonium sulfate precipitation of these antisera.

Localization of tropomyosin in mouse embryo fibroblasts.

Antiserum to chick skeletal muscle tropomyosin was used to localize tropomyosin in mouse embryo fibroblasts by the indirect fluorescein labeled antibody technique. Specific staining was observed cytoplasmic fibers, which extended out into the cell processes. The staining pattern in these cells is similar to that previously described by others for actin. This observation suggests that in fibroblasts tropomyosin, like actin, is localized in fibers in the cytoplasm.