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C Dingwall

Publications and source records attributed to C Dingwall.

At least 19 recordsLinked to original sources
PubMed1992

The nuclear membrane.

The nuclear membrane forms a major barrier within the cell, permitting levels of regulation not found in prokaryotes. The dynamics and diverse functions of the nuclear membrane and its associated structures are considered in this review. The role of the nuclear pore complex in selective transport across the nuclear membrane has been studied to a considerable degree; however, many crucial questions remain. Components of a signal transduction mechanism are associated with the nucleus, suggesting that nuclear functions may be influenced directly by this system. The involvement of the heat shock cognate protein Hsc70 in nuclear protein import is discussed, and a specific signal-presentation role for this protein is proposed.

Animals↗
PubMed1991

Two interdependent basic domains in nucleoplasmin nuclear targeting sequence: identification of a class of bipartite nuclear targeting sequence.

Point mutagenesis of the nuclear targeting sequence of nucleoplasmin has identified two interdependent basic domains. These are separated by 10 intervening "spacer" amino acids that tolerate point mutations and some insertions. Amino acids in both basic domains are required for nuclear targeting, and the transport defect of a mutation in one domain is amplified by a simultaneous mutation in the other. Therefore, these basic domains are interdependent. A strikingly similar motif of two clusters of basic residues is seen in the nuclear targeting sequence of Xenopus N1. It is also conserved in the related nucleolar protein NO38. Several other short sequences known to be necessary for nuclear targeting fall within a similar motif.

Amino Acid Sequence↗
PubMed1991

Transport across the nuclear envelope: enigmas and explanations.

The transport of molecules across the nuclear envelope plays a central role in the metabolism of the cell. Significant advances in three major areas highlight the limits of our current knowledge and point to the prospect of exciting future developments. Firstly, findings that ions and small proteins do not diffuse freely into the nucleus call into question the current views of nuclear envelope permeability. Secondly, indications that nuclear protein import can be regulated in conjunction with the cell cycle and development have been confirmed by the clear demonstration of regulated nuclear entry of specific transcription factors and morphogens. Thirdly, identification and characterisation of candidate receptor proteins indicates that the recognition of the nuclear targeting sequence occurs in the cytoplasm, suggesting that a different recognition event occurs at the nuclear pore.

Animals↗
PubMed1991

RNA binding by the tat and rev proteins of HIV-1.

HIV-1 tat protein binds specifically to HIV-1 TAR RNA. A Scatchard analysis of tat binding has shown that the purified protein forms a one-to-one complex with HIV-1 TAR RNA with a dissociation constant of Kd = 12 nM. Tat binding in vitro is dependent upon the presence of 3 non-base paired U residues which produce a 'bulge' in the TAR RNA stem-loop structure. Deletion of the uridine residues in the bulge or substitution with guanine residues produced RNAs with a 6 to 8-fold lower affinity than wild-type TAR. By contrast, mutations that alter the sequence of the 6 nucleotide-long loop at the tip of TAR RNA structure, and mutations which alter the sequence of the stem whilst preserving Watson-Crick base pairing, do not affect tat binding significantly. There is a direct correlation between the ability of tat to bind to TAR RNA and to activate HIV transcription. Viral LTRs encoding TAR sequences known to bind tat weakly, are not stimulated efficiently by tat in vivo. HIV-1 regulator of virion expression (rev) protein binds specifically to RNA transcripts containing the 223 nucleotide-long RRE sequence with an apparent dissociation constant of 1-3 nM. The minimum binding site for rev is a 'bubble' containing 2 G residues on one side and the sequence AGU on the other. Rev is able to bind efficiently to this restricted site in the context of the RRE sequence as well as in the context of a stable RNA duplex with a sequence unrelated to that found in the RRE.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1991

Nuclear targeting sequences--a consensus?

Nuclear targeting sequences are essential for the transport of proteins into the nucleus. The seven-amino-acid nuclear targeting sequence of the SV40 large T antigen has been regarded as the model; however, many nuclear targeting sequences appear to be more complex. We suggest in this review that, despite this diversity, a consensus bipartite motif can be identified.

Amino Acid Sequence↗
PubMed1990

HIV-1 regulator of virion expression (Rev) protein binds to an RNA stem-loop structure located within the Rev response element region.

HIV-1 Rev protein, purified from E. coli, binds specifically to an RNA transcript containing the 223 nucleotide long Rev response element (RRE) sequence. Rev binds to RRE in vitro with an apparent dissociation constant of 1 to 3 nM as determined by filter binding, gel mobility shift assays, or an immunoprecipitation assay using a monoclonal antibody specific for the Rev C-terminus. Antisense RRE sequences are bound by Rev with a 20-fold lower affinity than wild-type RRE sequences. The Rev-RRE complex forms even in the presence of a 10,000-fold molar excess of 16S rRNA, whereas formation of the low affinity antisense RRE-Rev complex is efficiently blocked by addition of excess 16S rRNA. A approximately 33 nucleotide fragment is protected from ribonuclease T1 digestion by the binding of Rev to RRE RNA, suggesting that Rev binds with high affinity to only a restricted region of the RRE. This protected fragment is unable to rebind Rev protein but has been mapped to a 71 nucleotide long Rev binding domain sequence that overlaps the protected fragment.

Amino Acid Sequence↗
PubMed1990

HIV-1 tat protein stimulates transcription by binding to a U-rich bulge in the stem of the TAR RNA structure.

The HIV-1 trans-activator protein, tat, is an RNA binding protein with a high affinity for a U-rich bulge near the tip of the stem in the RNA stem-loop structure encoded by the trans-activation responsive region (TAR). A Scatchard analysis of tat binding has shown that the purified protein forms a one-to-one complex with HIV-1 TAR RNA with a dissociation constant of Kd = 12 nM. Deletion of the uridine residues in the bulge or substitution with guanine residues produced RNAs with a 6- to 8-fold lower affinity than wild-type TAR. Introduction of a point mutation expected to destabilize base pairing in nearby residues of the TAR stem-loop structure reduced tat binding 10-fold. In contrast, mutations that alter the sequence of the six nucleotide long loop at the tip of TAR RNA structure, and mutations which alter the sequence of the stem whilst preserving Watson-Crick base pairing, do not affect tat binding significantly. There is a direct correlation between the ability of tat to bind to TAR RNA and to activate HIV transcription. Viral LTRs carrying TAR sequences encoding any of the mutations known to produce transcripts which bind tat weakly, are not stimulated efficiently by tat in vivo.

Base Sequence↗
PubMed1990

Nucleoplasmin: the archetypal molecular chaperone.

Nucleoplasmin was the first protein to be described as a molecular chaperone. Studies of nucleoplasmin have resulted in advances in two areas of cell biology. Firstly, the pathway of nucleosome assembly in Xenopus oocytes and eggs has been elucidated and is the only assembly pathway known in detail. Nucleosome assembly represents the major chaperoning function of nucleoplasmin. Secondly, nucleoplasmin has been used to elucidate the transport of proteins into the nucleus, revealing a selective entry mechanism for nuclear proteins, passage through the nuclear pore complex, and a two-step mechanism of transport. The properties and functions of nucleoplasmin are reviewed, together with other proteins which are related either structurally or functionally to nucleoplasmin.

Animals↗
PubMed1989

Human immunodeficiency virus 1 tat protein binds trans-activation-responsive region (TAR) RNA in vitro.

tat, the trans-activator protein for human immunodeficiency virus 1 (HIV-1), has been expressed in Escherichia coli from synthetic genes. Purified tat binds specifically to HIV-1 trans-activation-responsive region (TAR) RNA in gel-retardation, filter-binding, and immunoprecipitation assays. tat does not bind detectably to antisense TAR RNA sequences, cellular mRNA sequences, variant TAR RNA sequences with altered stem-loop structures, or TAR DNA.

Base Sequence↗
PubMed1989

Characterisation of the nuclear location sequence of Xenopus nucleoplasmin.

Each subunit of the Xenopus nucleoplasmin polypeptide possesses a single nuclear location sequence, the boundaries of which have been determined by deletion analysis. However, the sequence identified in this analysis is unable to locate pyruvate kinase to the cell nucleus. Further investigation revealed that while this sequence element is not sufficient for nuclear localisation, it is a necessary feature of slightly longer sequences which can locate pyruvate kinase to the cell nucleus. This unusual feature of the nucleoplasmin sequence suggests testable models for the interaction of this sequence with the transport mechanism, one of which is discussed here.

Amino Acid Sequence↗
PubMed1988

Nuclear protein migration involves two steps: rapid binding at the nuclear envelope followed by slower translocation through nuclear pores.

When injected into the cytoplasm of Vero cells, nucleoplasmin rapidly concentrates in a narrow layer around the nuclear envelope and then accumulates within the nucleus. Transport into the nucleus can be reversibly arrested at the perinuclear stage by metabolic inhibitors or by chilling. Nucleoplasmin-coated colloidal gold particles concentrate around the nuclear envelope of Vero cells or Xenopus oocytes, and by electron microscopy of oocytes appear to be associated with fibrils attached to nuclear pore complexes. Perinuclear accumulation is not observed for the nonmigrating nucleoplasmin core fragment or nonnuclear proteins. We propose two steps in nuclear migration of proteins: rapid binding around the nuclear envelope, possibly to pore-associated fibrils, followed by slower, energy-dependent translocation through nuclear pores.

Adenosine Triphosphate↗
PubMed1988

The nucleoplasmin nuclear location sequence is larger and more complex than that of SV-40 large T antigen.

The carboxy-terminal tail of nucleoplasmin, which specifies entry into the cell nucleus, contains four short sequences that are similar to previously identified nuclear location sequences. We show that none of these is able to locate chicken muscle pyruvate kinase to the cell nucleus. Deletion analysis was used to determine the limits of a nuclear location sequence and indicated that a 14-amino acid segment (RPAATKKAGQAKKK) should function as a minimal nuclear location sequence. When tested directly, however, this sequence was unable to locate pyruvate kinase to the cell nucleus. Restoration of three amino acids of nucleoplasmin sequence at either end of this sequence generated sequences that were able to locate pyruvate kinase to the cell nucleus. The 14-amino acid proposed minimal nuclear location sequence is present in the functional sequences, AVKRPAATKKAGQAKKK, RPAATKKAGQAKKKKLD, and the sequence AVKRPAATKKAGQAKKKKLD, which has additional amino acids at both ends. The minimal sequence element is therefore necessary but not sufficient for transport into the cell nucleus. This unusual feature of the nucleoplasmin nuclear location sequence suggests ways in which it could interact with the nuclear transport mechanism.

Amino Acid Sequence↗
PubMed1987

Chromosome replication in cell-free systems from Xenopus eggs.

Cell-free systems from eggs of the frog Xenopus laevis are able to perform most of the acts of eukaryotic chromosome replication in vitro. This now includes the crucial regulatory step of initiation, which had only been achieved for viral systems previously. Purified DNA or nuclei are able to initiate and complete semi-conservation replication in egg extracts in vitro (Blow & Laskey, Cell 47, 557-587 (1986). Replication does not require specialized DNA sequences either in vitro or in microinjected eggs, but in both systems large templates replicate more efficiently than small templates. In some cases replication can re-initiate, excluding the possibility that replication is primed by preexisting primers in the template preparations. When nuclei are replicated in vitro, only one round of replication is observed in a single incubation resembling the single round of replication observed for purified DNA after micro-injection. The mechanism that prevents re-initiation of replication within a single cell cycle is discussed and certain models are eliminated. Nucleosome assembly from histones and DNA has also been studied in cell-free systems from Xenopus eggs. Fractionation has led to the identification of two acidic proteins called nucleoplasmin and N1, which bind histones and transfer them to DNA. The sequences of both proteins have been determined by cDNA cloning and sequencing. Both proteins are found as complexes with histones in eggs.

Animals↗
PubMed1987

Nucleoplasmin cDNA sequence reveals polyglutamic acid tracts and a cluster of sequences homologous to putative nuclear localization signals.

Nucleoplasmin is the most abundant protein in the Xenopus oocyte nucleus. It is involved in histone storage and chromatin assembly and it has been used extensively to study the transport of proteins into the cell nucleus. We have isolated lambda gt11 phage containing nucleoplasmin cDNA and have determined the sequence of the entire protein coding region of 200 amino acids for one of the two genes. The translation product of the sp6 transcript of this cDNA has the same electrophoretic mobility as nucleoplasmin and is able to form pentamers. The protein sequence shows remarkable clusters of charged residues including a long polyglutamic acid tract which presumably constitutes the histone binding site. The short C-terminal domain which specifies nuclear entry contains four regions which are homologous to putative nuclear localization signals including two regions of homology to the nuclear migration signal of SV40 large T antigen.

Amino Acid Sequence↗
PubMed1986

Molecular characterization of a karyophilic, histone-binding protein: cDNA cloning, amino acid sequence and expression of nuclear protein N1/N2 of Xenopus laevis.

In the amphibian oocyte, most of the non-chromatin-bound histones are not free but form complexes with specific karyophilic proteins, the most prominent being nucleoplasmin and 'protein N1/N2'. Using antibodies against polypeptide N1 and N2 (Mr approximately 105,000 and approximately 110,000) we have isolated, from a Xenopus laevis ovary lambda gt11 expression library, several full length cDNA clones encoding one of the two closely related polypeptides N1 and N2 (these could not be distinguished by hybridization techniques). The amino acid sequence deduced from one of these clones (N1/N2, lambda 106.2) defines a polypeptide of mol. wt 64,774. The remarkably high difference between the value of Mr approximately 110,000 estimated from SDS-PAGE mobility and the true mol. wt has been found for (i) the cell protein, (ii) the polypeptide synthesized in vitro by transcription and translation and (iii) the fusion protein with beta-galactosidase expressed in Escherichia coli, indicating that the protein runs anomalously on SDS-PAGE. The amino and carboxy termini of the purified protein N1/N2 have been confirmed by direct amino acid sequencing of CNBr fragments. The amino acid sequence displays two glutamic acid-rich domains, which are probably involved in the interaction with the histones, and a putative nuclear targeting signal with high homology to that of the SV40 large T-antigen which is located near the carboxy terminus.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗