5' cleavage site in eukaryotic pre-mRNA splicing is determined by the overall 5' splice region, not by the conserved 5' GU

We have generated all possible single point mutations of the invariant 5' GT of the large beta-globin intron and determined their effect on splicing in vitro. None of the mutants prevented cleavage in the 5' splice region, but many reduced or abolished exon joining. The mutations GT----TT and GT----CT resulted in a shift of the 5' cleavage site on nucleotide upstream; in the case of the mutation GT----TT, this shift was reverted by a second site mutation within the 5' splice region. Our results suggest that the 5' cleavage site is determined not by the conserved GU sequence but by the 5' splice region as a whole, most probably via base-pairing to the 5' end of the U1 snRNA.     

cDNA structures and regulation of two interferon-induced human Mx proteins.

Human cells treated with interferon synthesize two proteins that exhibit high homology to murine Mx1 protein, which has previously been identified as the mediator of interferon-induced cellular resistance of mouse cells against influenza viruses. Using murine Mx1 cDNA as a hybridization probe, we have isolated cDNA clones originating from two distinct human Mx genes, designated MxA and MxB. In human fibroblasts, expression of MxA and MxB is strongly induced by alpha interferon (IFN-alpha), IFN-beta, Newcastle disease virus, and, to a much lesser extent, IFN-gamma, MxA and MxB proteins have molecular masses of 76 and 73 kilodaltons, respectively, and their sequences are 63% identical. A comparison of human and mouse Mx proteins revealed that human MxA and mouse Mx2 are the most closely related proteins, showing 77% sequence identity. Near their amino termini, human and mouse Mx proteins contain a block of 53 identical amino acids and additional regions of very high sequence similarity. These conserved sequences are also present in a double-stranded RNA-inducible fish gene, which suggests that they may constitute a functionally important domain of Mx proteins. In contrast to mouse Mx1 protein, which accumulates in the nuclei of IFN-treated mouse cells, the two human Mx proteins both accumulate in the cytoplasm of IFN-treated cells.     

Organization of the murine Mx gene and characterization of its interferon- and virus-inducible promoter.

Specific resistance of Mx+ mice to influenza virus is due to the interferon (IFN)-induced protein Mx. The Mx gene consists of 14 exons that are spread over at least 55 kilobase pairs of DNA. Surprisingly, the Mx gene promoter is induced as efficiently by Newcastle disease virus as it is by IFN. The 5' boundary of the region required for maximal induction by both IFN and Newcastle disease virus is located about 140 base pairs upstream of the cap site. This region contains five elements of the type GAAANN, which occurs in all IFN- and virus-inducible promoters. The consensus sequence purine-GAAAN(N/-)GAAA(C/G)-pyrimidine is found in all IFN-inducible promoters.     

Disassembly of in vitro formed lamin head-to-tail polymers by CDC2 kinase.

The nuclear lamina is an intermediate filament-type network underlying the inner nuclear membrane. At the onset of mitosis it depolymerizes, presumably in response to phosphorylation of the lamin proteins. Recently, cdc2 kinase, a major regulator of the eukaryotic cell cycle, was shown to induce lamina depolymerization when incubated with isolated nuclei. Here, we have analysed the structural consequences of lamin phosphorylation by cdc2 kinase using lamin head-to-tail polymers reconstituted in vitro from bacterially expressed chicken lamin B2 protein as a substrate. The effects of phosphorylation were monitored by both a pelleting assay and electron microscopy. We show that lamin B2 head-to-tail polymers disassemble in response to phosphorylation of specific sites that are phosphorylated also during mitosis in vivo. These sites are located within SP/TP motifs N- and C-terminal to the central alpha-helical rod domain of lamin proteins. Subsequent dephosphorylation of these sites by purified phosphatase 1 allows reformation of lamin head-to-tail polymers. The relative importance of N- and C-terminal phosphorylation sites for controlling the assembly state of nuclear lamins was assessed by mutational analysis. Polymers formed of lamin proteins carrying mutations in the C-terminal phosphoacceptor motif could still be disassembled by cdc2 kinase. In contrast, a single point mutation in the N-terminal site (Ser16----Ala) rendered head-to-tail polymers resistant to disassembly. These results emphasize the importance of the N-terminal end domain for lamin head-to-tail polymerization in vitro, and they demonstrate that phosphorylation-dephosphorylation is sufficient to control the longitudinal assembly of lamin B2 dimers.     

Inactivation of nonsense suppressor transfer RNA genes in Schizosaccharomyces pombe. Intergenic conversion and hot spots of mutation

Intergenic conversion is a mechanism for the concerted evolution of repeated DNA sequences. A new approach for the isolation of intergenic convertants of serine tRNA genes in the yeast Schizosaccharomyces pombe is described. Contrary to a previous scheme, the intergenic conversion events studied in this case need not result in functional tRNA genes. The procedure utilizes crosses of strains that are homozygous for an active UGA suppressor tRNA gene, and the resulting progeny spores are screened for loss of suppressor activity. In this way, intergenic convertants of a tRNA gene are identified that inherit varying stretches of DNA sequence from either of two other tRNA genes. The information transferred between genes includes anticodon and intron sequences. Two of the three tRNA genes involved in these information transfers are located on different chromosomes. The results indicate that intergenic conversion is a conservative process. No infidelity is observed in the nucleotide sequence transfers. This provides further evidence for the hypothesis that intergenic conversion and allelic conversion are the result of the same molecular mechanism. The screening procedure for intergenic revertants also yields spontaneous mutations that inactivate the suppressor tRNA gene. Point mutations and insertions of A occur at various sites at low frequency. In contrast, A insertions at one specific site occur with high frequency in each of the three tRNA genes. This new type of mutation hot spot is found also in vegetative cells.     

Polymorphism of reconstituted human epidermal keratin filaments: determination of their mass-per-length and width by scanning transmission electron microscopy (STEM)

We have determined the mass-per-length (MPL) and the width of unstained freeze-dried reconstituted human epidermal keratin filaments by scanning transmission electron microscopy (STEM). Filaments were reassembled from keratins extracted from four different sources: cultured human epidermal cells (CHEC), human callus (CAL), and the living layers (LL) and stratum corneum (SC) of normal human epidermis. MPL histograms of all four keratin filament types could be fitted by a superposition of two or three Gaussians, with their respective major peaks located between 17 and 20 kDa/nm. We interpreted the multiple MPL peaks to represent different polymorphic forms of the reconstituted filaments. The number of subunits per filament cross section calculated from MPL peak positions, average subunit molecular weight, and an axial repeat of the subunits within the filament of 46.5 nm revealed an average difference between polymorphic variants of 7.5 +/- 0.9 subunits. These data suggest that reconstituted human epidermal keratin filaments are made of two to four 8-stranded "protofibrils" (i.e., made of two laterally aggregated 4-stranded protofilaments), in agreement with earlier observations. The average widths of unstained freeze-dried keratin filaments were larger than those of negatively stained filaments: 12.6 nm (9.6 nm) for CHEC, 12.3 nm (9.7 nm) for CAL, 11.6 nm (8.3 nm) for LL, and 11.3 nm (7.9 nm) for SC keratin filaments, with the values in brackets corresponding to negatively stained samples. Assuming the MPL to be proportional to the square of the filament width, there is a good correlation between the MPL and width measurements both for filaments within a given type as well as among those reconstituted from different types of keratin extracts.     

Association of kidney and parotid Na+, K(+)-ATPase microsomes with actin and analogs of spectrin and ankyrin

Kidney Na+,K(+)-ATPase has been recently shown to bind erythroid ankyrin and to colocalize with ankyrin at the basolateral cell surface of kidney epithelial cells. These observations suggest that Na+,K(+)-ATPase is linked via ankyrin to the spectrin/actin-based membrane cytoskeleton. In the present study we show that Na+,K(+)-ATPase and analogs of spectrin, ankyrin and actin copurify from detergent extracts of pig kidney and parotid gland membranes. Actin, spectrin and ankyrin were extracted from purified Na+,K(+)-ATPase microsomes at virtually identical conditions as their counterparts from the erythrocyte membrane, i.e., 1 mM EDTA (spectrin, actin) and 1 M KCl (ankyrin). Visualization of the stripped proteins by rotary shadowing revealed numerous elongated spectrin-like dimers (100 nm) and tetramers (215 nm), a fraction of which (17%) was associated with globular (10 nm) ankyrin-like particles. Like erythrocyte ankyrin, kidney ankyrin was cleaved into a soluble 72 kDa fragment and a membrane-bound 90 kDa fragment. Consistent with our previous immunocytochemical findings on the pig kidney, Na+,K(+)-ATPase and ankyrin were found to be colocalized at the basolateral plasma membrane of striated ducts and acini of the pig parotid gland. The present findings confirm and extend the recently proposed concept that in polarized epithelial cells Na+,K(+)-ATPase may serve as major attachment site for the spectrin-based membrane cytoskeleton to the basolateral cell domain. Connections of integral membrane proteins to the cytoskeleton may help to place these proteins at specialized domains of the cell surface and to prevent them from endocytosis.     

Identification of different roles for RanGDP and RanGTP in nuclear protein import.

The importin-alpha/beta heterodimer and the GTPase Ran play key roles in nuclear protein import. Importin binds the nuclear localization signal (NLS). Translocation of the resulting import ligand complex through the nuclear pore complex (NPC) requires Ran and is terminated at the nucleoplasmic side by its disassembly. The principal GTP exchange factor for Ran is the nuclear protein RCC1, whereas the major RanGAP is cytoplasmic, predicting that nuclear Ran is mainly in the GTP form and cytoplasmic Ran is in the GDP-bound form. Here, we show that nuclear import depends on cytoplasmic RanGDP and free GTP, and that RanGDP binds to the NPC. Therefore, import might involve nucleotide exchange and GTP hydrolysis on NPC-bound Ran. RanGDP binding to the NPC is not mediated by the Ran binding sites of importin-beta, suggesting that translocation is not driven from these sites. Consistently, a mutant importin-beta deficient in Ran binding can deliver its cargo up to the nucleoplasmic side of the NPC. However, the mutant is unable to release the import substrate into the nucleoplasm. Thus, binding of nucleoplasmic RanGTP to importin-beta probably triggers termination, i.e. the dissociation of importin-alpha from importin-beta and the subsequent release of the import substrate into the nucleoplasm.     

Identification and Characterization of a cDNA and the Structural Gene Encoding the Mouse Epithelial Membrane Protein-1

The PMP22/EMP/MP20 gene family includes four closely related proteins, peripheral myelin protein-22 (PMP22), epithelial membrane protein-1 (EMP-1), epithelial membrane protein-2 (EMP-2), and epithelial membrane protein-3 (EMP-3), which share amino acid identities ranging from 33 to 43%. In addition, the lens-specific membrane protein MP20 represents a more distant relative. Functionally, this family of proteins is likely to play important roles in the control of cell proliferation, cell differentiation, and cell death. In particular, mutations affecting thePMP22gene are responsible for various hereditary peripheral neuropathies in humans and mice. We report the isolation and characterization of a mouse EMP-1 cDNA and the correspondingemp-1gene. Mouse EMP-1 displays 93% amino acid identity to rat EMP-1 and 39% identity to mouse PMP22. The cDNA-predicted EMP-1 protein contains four putative membrane-associated domains and can beN-linked glycosylatedin vitro.EMP-1 is encoded by a single-copy gene with the positions of introns exactly conserved betweenemp-1andPMP22,corroborating the hypothesis that both genes belong to the same family. Computer-predicted structural domains of EMP-1 are partially mirrored by the exon/intron structure ofemp-1.Most interestingly, exon 4, which covers the potential second transmembrane domain, a small intracellular loop, and half of the third transmembrane domain, encodes the most highly conserved regions between the EMP-1 and PMP22 proteins and is also remarkably conserved in the MP20 gene, indicating some shared functional significance for this module in the PMP22/EMP/MP20 family.