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PubMed1994

The genomic structure of an insertional mutation in the dystonia musculorum locus.

We have previously identified a line of transgenic mice, Tg4, in which an hsp68-lacZ hybrid gene has inserted into the dystonia musculorum (dt) locus on chromosome 1. We have confirmed the localization of the Tg4 integration site to the proximal region of mouse chromosome 1 by interspecific backcross analysis. One end of the integration complex has been cloned and we have used single-copy probes from the flanking region to screen a mouse genomic library. Several overlapping lambda phage clones have been isolated and arranged into a contig spanning 75 kb of genomic DNA. Probes from the genomic contig have enabled us to characterize the wildtype and Tg4 loci. We report that the integration of the transgene was accompanied by a deletion of 45 kb of host genomic sequences with no other detectable rearrangement in the Tg4 genome.

Animals↗

PubMed1995

Murine collagen intron-binding factor I (CIBF-I) is the same protein as transcription factor Oct-1.

The recognition sequence (CIB) of collagen intron-binding factor I (CIBF-I) loosely resembles the consensus octamer-binding motif (OCT). In the present study we investigate whether CIBF-I is actually the OCT-binding protein, Oct-1. Electrophoretic mobility-shift assays (EMSA) demonstrate that a consensus OCT motif effectively competes for CIBF-I binding. CIBF-I and Oct-1 complexes display similar EMSA characteristics, and both factors are detected in nuclear extracts of five different cell types. In addition, pre-incubation of nuclear extracts with antiserum directed against the POU domain of Oct-1 inhibits CIBF-I complex formation. Finally, DNA transfection experiments demonstrate that a single copy of the CIB site is sufficient to stimulate transcription from the SV40 early promoter in NIH 3T3 cells. These results suggest that CIBF-I is the ubiquitously distributed OCT-binding protein, Oct-1, and represent the first report that an octamer-binding protein contributes to the transcriptional activity of a collagen-encoding gene.

Animals↗

PubMed2005

Architecture of the ribosome-channel complex derived from native membranes.

The mammalian Sec61 complex forms a protein translocation channel whose function depends upon its interaction with the ribosome and with membrane proteins of the endoplasmic reticulum (ER). To study these interactions, we determined structures of "native" ribosome-channel complexes derived from ER membranes. We find that the ribosome is linked to the channel by seven connections, but the junction may still provide a path for domains of nascent membrane proteins to move into the cytoplasm. In addition, the native channel is significantly larger than a channel formed by the Sec61 complex, due to the presence of a second membrane protein. We identified this component as TRAP, the translocon-associated protein complex. TRAP interacts with Sec61 through its transmembrane domain and has a prominent lumenal domain. The presence of TRAP in the native channel indicates that it may play a general role in translocation. Crystal structures of two Sec61 homologues were used to model the channel. This analysis indicates that there are four Sec61 complexes and two TRAP molecules in each native channel. Thus, we suggest that a single Sec61 complex may form a conduit for translocating polypeptides, while three copies of Sec61 play a structural role or recruit accessory factors such as TRAP.

Animals↗

PubMed2004

The mechanism of sister chromatid cohesion.

Each of our cells inherit their genetic information in the form of chromosomes from a mother cell. In order that we obtain the full genetic complement, cells need to ensure that replicated chromosomes are accurately split and distributed during cell division. Mistakes in this process lead to aneuploidies, cells with supernumerous or missing chromosomes. Most aneuploid human embryos are not viable, and if they are, they develop severe birth defects. Aneuploidies later in human life are frequently found associated with the development of malignant cancer. DNA replication during S-phase is linked to segregation of the sister copies in mitosis by sister chromatid cohesion. A chromosomal protein complex, cohesin, holds replicated sister DNA strands together after their synthesis. This allows pairs of replication products to be recognised by the spindle apparatus in mitosis for segregation into opposite direction. At anaphase onset, cohesin is destroyed by a site-specific protease, separase. Here I review what we have learned about the molecular mechanism of sister chromatid cohesion. Cohesin forms a large proteinaceous ring that may hold sister chromatids by encircling and topological trapping. To understand how cohesin links newly synthesised replication products, biochemical assays to study the enzymology of cohesin will be required.

Animals↗

PubMedDate not supplied

An insect antibacterial peptide-based drug delivery system.

The ability of the short, proline-rich native antibacterial peptides to penetrate bacterial and host cells suggests the utility of these transport systems in delivering peptidic cargo into cells. We studied the uptake of pyrrhocoricin and its most potent dimeric analogue by bacteria as well as human dendritic cells and fibroblasts. Native pyrrhocoricin entered the susceptible organism Escherichia coli very efficiently and the nonsusceptible bacterium Staphylococcus aureus to a significant degree. The antibacterial peptide also penetrated human monocyte-derived dendritic cells. It failed, however, to enter fibroblasts, whereas the designer analogue Pip-pyrr-MeArg dimer penetrated all the cell types that were studied. When glucoincretin hormone Glp-1 fragment 7-36 was cosynthesized with the dimer, the antibacterial peptide derivative lost its ability to cross the bacterial membrane layer. In contrast, a chimera of the Pip-pyrr-MeArg dimer and two copies of a shorter (nine residues) class I major histocompatibility complex epitope successfully entered bacterial and mammalian cells. While the Pip-pyrr-MeArg dimer was not immunogenic when inoculated into mice, the chimera elicited a strong cytotoxic T-cell response, indicating the maintenance of the antigenic integrity of the cargo in the peptide conjugate. The chimera when tested for its immunological properties activated human dendritic cells significantly more strongly than any of the two independent fragments alone, yet lacked mammalian cell toxicity. These results confirm the utility of designed pyrrhocoricin analogues for delivery of peptidic cargo across cell membranes in general, and their potential as carriers for epitope-based vaccines in particular.

Amino Acid Sequence↗

PubMed2001

Purification and characterization of the membrane-bound complex of an ABC transporter, the histidine permease.

The bacterial histidine permease, an ABC transporter, from Salmonella typhimurium is composed of a membrane-bound complex, HisQMP2, comprising two hydrophobic subunits (HisQ and HisM), two copies of an ATP-hydrolyzing subunit, HisP, and a soluble receptor, HisJ. We describe the purification and characterization of HisQMP2 using a 6-histidines extension at the carboxy terminus of HisP [HisQMP2(his6)]. The purification is rapid and effective, giving a seven-fold purification with a yield of 85 and 98% purity. Two procedures are described differing in the detergent used (decanoylsucrose and octylglucoside, respectively) and in the presence of phospholipid. HisQMP2(his6) has ATPase and transport activities upon reconstitution into proteoliposomes (PLS). HisQMP2(his6) has a low level ATPase activity (intrinsic activity), which is stimulated to a different extent by the receptor--liganded and unliganded. Its pH optimum is 7.8-8.0, it requires a cation for activity and it displays cooperativity for ATP. The effect of various ATP analogs was analyzed. Determination of the molecular size of HisQMP2(his6) indicates that it is a monomer. The permeability properties of two kinds of reconstituted PLS preparations are described.

ATP-Binding Cassette Transporters↗

PubMed1972

A new synthetic RNA-dependent DNA polymerase from human tissue culture cells (HeLa-fibroblast-synthetic oligonucleotides-template-purified enzymes).

Two DNA polymerases that can copy synthetic RNA polymers are present in human tissue culture cells. These enzymes which have each been purified about 500-fold, are present in both HeLa cells, which are derived from a cervical carcinoma, and in WI-38 cells, a normal diploid strain originating from human embryonic lung tissue. These synthetic RNA-dependent DNA polymerases are identified by their ability to copy efficiently the ribo strand of synthetic oligonucleotide-homopolymer complexes, and differ in this respect from the known DNA-dependent DNA polymerases found in HeLa cells. The template requirements of these new DNA polymerases resemble that of the RNA-dependent DNA polymerases of the RNA tumor-viruses.

Cell Line↗

PubMed2003

Regulation of the sequential processing of Semliki Forest virus replicase polyprotein.

The replication of most positive-strand RNA viruses and retroviruses is regulated by proteolytic processing. Alphavirus replicase proteins are synthesized as a polyprotein, called P1234, which is cleaved into nsP1, nsP2, nsP3, and nsP4 by the carboxyl-terminal protease domain of nsP2. The cleavage intermediate P123+nsP4 synthesizes minus-strand copies of the viral RNA genome, whereas the completely processed complex is required for plus-strand synthesis. To understand the mechanisms responsible for this sequential proteolysis, we analyzed in vitro translated Semliki Forest virus polyproteins containing noncleavable processing sites or various deletions. Processing of each of the three sites in vitro required a different type of activity. Site 3/4 was cleaved in trans by nsP2, its carboxyl-terminal fragment Pro39, and by all polyprotein proteases. Site 1/2 was cleaved in cis with a half-life of about 20-30 min. Site 2/3 was cleaved rapidly in trans but only after release of nsP1 from the polyprotein exposing an "activator" sequence present in the amino terminus of nsP2. Deletion of amino-terminal amino acids of nsP2 or addition of extra amino acid residues to its amino terminus specifically inhibited the protease activity that processes the 2/3 site. This sequence of delayed processing of P1234 would explain the accumulation of P123 plus nsP4, the early short-lived minus-strand replicase. The polyprotein stage would allow correct assembly and membrane association of the RNA-polymerase complex. Late in infection free nsP2 would cleave at site 2/3 yielding P12 and P34, the products of which, nsP1-4, are distributed to the plasma membrane, nucleus, cytoplasmic aggregates, and proteasomes, respectively.

Cysteine Endopeptidases↗

PubMed1995

NUP82 is an essential yeast nucleoporin required for poly(A)+ RNA export.

We have isolated and characterized the gene encoding a novel essential nucleoporin of 82 kD, termed NUP82. Indirect immunofluorescence of cells containing an epitope tagged copy of the NUP82 localized it to the nuclear pore complex (NPC). Primary structure analysis indicates that the COOH-terminal 195 amino acids contain a putative coiled-coil domain. Deletion of the COOH-terminal 87 amino acids of this domain causes slower cell growth; deletion of the COOH-terminal 108 amino acids results in slower growth at 30 degrees C and lethality at 37 degrees C. Cells in which the last 108 amino acids of NUP82 have been deleted, when shifted to 37 degrees C, do not display any gross morphological defects in their nuclear pore complexes or nuclear envelopes. They do, however, accumulate poly(A)+ RNA in their nuclei at 37 degrees C. We propose that NUP82 acts as a linker to tether nucleoporins directly involved in nuclear transport to pore scaffolding via its coiled-coil domain.

Amino Acid Sequence↗

PubMed2000

The role of the tethering proteins p115 and GM130 in transport through the Golgi apparatus in vivo.

Biochemical data have shown that COPI-coated vesicles are tethered to Golgi membranes by a complex of at least three proteins: p115, giantin, and GM130. p115 binds to giantin on the vesicles and to GM130 on the membrane. We now examine the function of this tethering complex in vivo. Microinjection of an N-terminal peptide of GM130 or overexpression of GM130 lacking this N-terminal peptide inhibits the binding of p115 to Golgi membranes. Electron microscopic analysis of single microinjected cells shows that the number of COP-sized transport vesicles in the Golgi region increases substantially, suggesting that transport vesicles continue to bud but are less able to fuse. This was corroborated by quantitative immunofluorescence analysis, which showed that the intracellular transport of the VSV-G protein was significantly inhibited. Together, these data suggest that this tethering complex increases the efficiency with which transport vesicles fuse with their target membrane. They also provide support for a model of mitotic Golgi fragmentation in which the tethering complex is disrupted by mitotic phosphorylation of GM130.

Animals↗

PubMed2000

Composing life.

Textbooks often assert that life began with specialized complex molecules, such as RNA, that are capable of making their own copies. This scenario has serious difficulties, but an alternative has remained elusive. Recent research and computer simulations have suggested that the first steps toward life may not have involved biopolymers. Rather, non-covalent protocellular assemblies, generated by catalyzed recruitment of diverse amphiphilic and hydrophobic compounds, could have constituted the first systems capable of information storage, inheritance and selection. A complex chain of evolutionary events, yet to be deciphered, could then have led to the common ancestors of today's free-living cells, and to the appearance of DNA, RNA and protein enzymes.

Biological Evolution↗

PubMed1997

Roles of the influenza virus polymerase and nucleoprotein in forming a functional RNP structure.

Influenza virus transcription and replication is performed by ribonucleoprotein particles (RNPs). They consist of an RNA molecule covered with many copies of nucleoprotein (NP) and carry a trimeric RNA polymerase complex. RNA modification analysis and electron microscopy performed on native RNPs suggest that the polymerase forms a complex with both conserved viral RNA (vRNA) ends, whereas NP binding exposes the RNA bases to the solvent. After chemical removal of the polymerase, the bases at the vRNA extremities become reactive to modification and the vRNPs behave as structures with free ends, as judged from the observation of salt-induced conformational changes by electron microscopy. The vRNA appears to be completely single-stranded in polymerase-free RNPs despite a partial, inverted complementarity of the vRNA ends. The absence of a stable double-stranded panhandle structure in polymerase-free RNPs has important implications for the mechanism of viral transcription and the switch from transcription to replication.

Animals↗

PubMed2000

The sno gene, which encodes a component of the histone deacetylase complex, acts as a tumor suppressor in mice.

The Ski and Sno oncoproteins are components of a macromolecular complex containing the co-repressor N-CoR/SMRT, mSin3 and histone deacetylase. This complex has been implicated in the transcriptional repression exerted by a number of repressors including nuclear hormone receptors and Mad. Further more, Ski and Sno negatively regulate transforming growth factor-beta (TGF-beta) signaling by recruiting this complex to Smads. Here we show that loss of one copy of sno increases susceptibility to tumorigenesis in mice. Mice lacking sno died at an early stage of embryogenesis, and sno was required for blastocyst formation. Heterozygous (sno(+/-)) mice developed spontaneous lymphomas at a low frequency and showed an increased level of tumor formation relative to wild-type mice when challenged with a chemical carcinogen. sno(+/-) embryonic fibroblasts had an increased proliferative capacity and the introduction of activated Ki-ras into these cells resulted in neoplastic transformation. The B cells, T cells and embryonic fibroblasts of sno(+/-) mice had a decreased sensitivity to apoptosis or cell cycle arrest. These findings demonstrate that sno acts as a tumor suppressor at least in some types of cells.

Animals↗

PubMed2004

Dynamics of photosystem II: a proteomic approach to thylakoid protein complexes.

Oxygenic photosynthesis produces various radicals and active oxygen species with harmful effects on photosystem II (PSII). Such photodamage occurs at all light intensities. Damaged PSII centres, however, do not usually accumulate in the thylakoid membrane due to a rapid and efficient repair mechanism. The excellent design of PSII gives protection to most of the protein components and the damage is most often targeted only to the reaction centre D1 protein. Repair of PSII via turnover of the damaged protein subunits is a complex process involving (i) highly regulated reversible phosphorylation of several PSII core subunits, (ii) monomerization and migration of the PSII core from the grana to the stroma lamellae, (iii) partial disassembly of the PSII core monomer, (iv) highly specific proteolysis of the damaged proteins, and finally (v) a multi-step replacement of the damaged proteins with de novo synthesized copies followed by (vi) the reassembly, dimerization, and photoactivation of the PSII complexes. These processes will shortly be reviewed paying particular attention to the damage, turnover, and assembly of the PSII complex in grana and stroma thylakoids during the photoinhibition-repair cycle of PSII. Moreover, a two-dimensional Blue-native gel map of thylakoid membrane protein complexes, and their modification in the grana and stroma lamellae during a high-light treatment, is presented.

Light↗

PubMed1990

Two separable promoters control different aspects of expression of a Dictyostelium gene.

A single copy Dictyostelium gene was dissected and elements responsible for its complex pattern of regulation were defined by transcript analysis of gene fusions. Two overlapping promoters responsible for the transcription of an 'L' and an 'S' mRNA could be defined. Further dissection of the P8A7 L promoter resulted in the identification of a sequence necessary for stress induction and an element required for vegetative expression. The P8A7 S promoter could be reduced to 449 bp which were sufficient for expression in developing cells. The sequence element required for this transcriptional activity was shown to reside in a 51 bp fragment. Our results show that differential expression of the P8A7 gene is mediated by two independently functioning promoters which, however, share some regulatory elements. A third nuclear RNA species 'P' was due to the stress-sensitivity of the 3' processing signal.

Amino Acid Sequence↗

PubMed1996

Immunoreactivity and conformation of the P-P-G-M-R-P-P repetitive epitope of the Sm autoantigen.

Anti-Sm antibodies are usually considered highly specific for systemic lupus erythematosus (SLE), while anti-U1 RNP antibodies are found in high titers in patients with mixed connective tissue disease (MCTD). The sequence P1-P-G-M-R-P-P7, present in three copies in the Sm (U1-U6 RNA-protein complex) autoantigen, is an important functional domain of the antigenic determinants. The immunoreactivity of this proline-rich repetitive epitope was investigated by testing sera with various autoantibody specificities for reactivity against this epitope, as well as its conformational properties by means of 1D and 2D 1HNMR spectroscopy. It was found that the P-P-G-M-R-P-P epitope is recognized mainly by anti-U1RNP and/or anti-Sm positive sera, but also by anti-Ro(SSA) (hY1RNA-protein complex) and anti-La(SSB) (hY1RNA-protein complex) positive sera, although these sera are negative for anti-U1RNP and anti-Sm. Conformational analysis of the proline-rich epitope in DMSO-d6 solution obtained from lyophilized aqueous solution at pH 5 showed the presence of at least three conformers. The main conformer A (62%) is stabilized by an ionic interaction between the guanidinium and the C-terminal carboxylate groups, and the Pro6-Pro7 peptide bond adopts the cis form. A type II beta-turn is also present in the N-terminal sequence (Pro1-Pro-Gly-Met4-) of this conformer. Conformer B (21%) is also stabilized by a similar ionic interaction, as in conformer A, while the NMR data indicate the absence of a folded structure in the N-terminal tetrapeptide of this conformer. Conformer C (17%) adopts a completely extended structure. The multiple conformers of the P-P-G-M-R-P-P may offer some explanation for the reactivity of sera with various autoantibody specificities against this epitope.

Amino Acid Sequence↗

PubMed2006

Structure of Kluyveromyces lactis subtelomeres: duplications and gene content.

We have constructed a map of the duplicated regions of Kluyveromyces lactis subtelomeres. Seven out of 12 subtelomeres contain an almost identical 9 kb long segment starting from the end. This segment is bordered by a long terminal repeat element. Two of the subtelomeres share sequence similarity that extends over a total of 20 kb. The other subtelomeres also contain duplicated regions of 1-6 kb. Nonduplicated regions contain unique genes and genes from paralog families. All duplicated segments are in the same orientation with respect to the telomere, probably as a result of genetic exchange. We map the only two copies of retrotransposons in the genome, in subtelomeres. Low-complexity gene sequences that encode threonine- and serine-rich peptides are associated with the subtelomeres of K. lactis, as in Saccharomyces cerevisiae. The ubiquity of these sequences in hemiascomycete genomes, and the propensity they have to encode proteins with extracellular localization, make these genes ideal candidates for fast evolving 'contingency' genes involved in the adaptation of a species to its environment.

Amino Acid Sequence↗

PubMed2003

Single-copy green fluorescent protein gene fusions allow accurate measurement of Salmonella gene expression in vitro and during infection of mammalian cells.

We developed a reliable and flexible green fluorescent protein (GFP)-based system for measuring gene expression in individual bacterial cells. Until now, most systems have relied upon plasmid-borne gfp gene fusions, risking problems associated with plasmid instability. We show that a recently developed GFP variant, GFP+, is suitable for assessing bacterial gene expression. Various gfp+ transcriptional fusions were constructed and integrated as single copies into the chromosome of Salmonella enterica serovar Typhimurium. A comparison of the expression levels of proU-lacZ and proU-gfp+ fusions showed that GFP+ reported proU activity in individual Salmonella cells as accurately as beta-galactosidase reported activity for entire populations. The single-copy gfp+ fusions were ideal for monitoring up- and downregulation of Salmonella virulence genes. We discovered that in vitro induction of the SPI1gene prgH occurs only in a portion of the population and that the proportion varies with the growth phase. We determined the level of expression of the SPI2 gene ssaG in bacteria released from murine macrophages. Our results demonstrate for the first time that single-copy GFP+ fusions reliably report gene expression in simple and complex environments. This approach promises to allow accurate measurement of gene expression in individual bacteria during animal infection.

Animals↗