Biomedical subjects

C Macaulay

Publications and source records attributed to C Macaulay.

12 recordsLinked to original sources

PubMed1996

Assembly of the nuclear pore: biochemically distinct steps revealed with NEM, GTP gamma S, and BAPTA.

A key event in nuclear formation is the assembly of functional nuclear pores. We have used a nuclear reconstitution system derived from Xenopus eggs to examine the process of nuclear pore assembly in vitro. With this system, we have identified three reagents which interfere with nuclear pore assembly, NEM, GTP gamma S, and the Ca++ chelator, BAPTA. These reagents have allowed us to determine that the assembly of a nuclear pore requires the prior assembly of a double nuclear membrane. Inhibition of nuclear vesicle fusion by pretreatment of the membrane vesicle fraction with NEM blocks pore complex assembly. In contrast, NEM treatment of already fused double nuclear membranes does not block pore assembly. This indicates that NEM inhibits a single step in pore assembly--the initial fusion of vesicles required to form a double nuclear membrane. The presence of GTP gamma S blocks pore assembly at two distinct steps, first by preventing fusion between nuclear vesicles, and second by blocking a step in pore assembly that occurs on already fused double nuclear membranes. Interestingly, when the Ca2+ chelator BAPTA is added to a nuclear assembly reaction, it only transiently blocks nuclear vesicle fusion, but completely blocks nuclear pore assembly. This results in the formation of a nucleus surrounded by a double nuclear membrane, but devoid of nuclear pores. To order the positions at which GTP gamma S and BAPTA interfere with pore assembly, a novel anchored nuclear assembly assay was developed. This assay revealed that the BAPTA-sensitive step in pore assembly occurs after the second GTP gamma S-sensitive step. Thus, through use of an in vitro nuclear reconstitution system, it has been possible to biochemically define and order multiple steps in nuclear pore assembly.

Animals↗

PubMed1995

Differential mitotic phosphorylation of proteins of the nuclear pore complex.

During each cell cycle, the nucleus of higher eukaryotes undergoes a dramatic assembly and disassembly. These events can be faithfully reproduced in vitro using cell-free extracts derived from Xenopus eggs. Such extracts contain three major N-acetylglucosaminylated proteins, p200, p97, and p60. All three become assembled into reconstituted nuclear pores. Here we show that p200, p97, and p60 exist in eggs in soluble high molecular mass complexes of 1000, 450, and 600 kDA, respectively. The bulk of p60 is stably associated with proteins of 58 and 54 kDa, while p200 is associated with a fraction of p60 in a separate complex lacking p58 and p54. Upon examining the behavior of these proteins in the cell cycle, we find that p200 and p97 are highly phosphorylated at mitosis, both in vivo and in vitro. Moreover, in extracts that cycle between interphase and mitosis, p200 and p97 are specifically phosphorylated at mitosis. Corresponding with their mitotic phosphorylation, both p200 and p97 are specific substrates for purified mitotic Cdc2 kinase, whereas nucleoporin p60 is not. Analysis indicates that the size of the complexes containing the pore N-acetylglucosamine glycoproteins does not change during mitosis, suggesting that such complexes represent stable multicomponent modules into which the nucleus disassembles at mitosis.

Animals↗

PubMed1995

Reconstituted nuclei depleted of a vertebrate GLFG nuclear pore protein, p97, import but are defective in nuclear growth and replication.

Xenopus egg extracts provide a powerful system for in vitro reconstitution of nuclei and analysis of nuclear transport. Such cell-free extracts contain three major N-acetylglucosaminylated proteins: p200, p97, and p60. Both p200 and p60 have been found to be components of the nuclear pore. Here, the role of p97 has been investigated. Xenopus p97 was isolated and antisera were raised and affinity purified. Immunolocalization experiments indicate that p97 is present in a punctate pattern on the nuclear envelope and also in the nuclear interior. Peptide sequence analysis reveals that p97 contains a GLFG motif which defines a family of yeast nuclear pore proteins, as well as a peptide that is identical at 11/15 amino acids to a specific member of the GLFG family, NUP116. An additional peptide is highly homologous to a second sequence found in NUP116 and other members of the yeast GLFG family. A monoclonal antibody to the GLFG domain cross-reacts with a major Xenopus protein of 97 kD and polyclonal antiserum to p97 recognizes the yeast GLFG nucleoporin family. The p97 antiserum was used to immunodeplete Xenopus egg cytosol and p97-deficient nuclei were reconstituted. The p97-depleted nuclei remained largely competent for nuclear protein import. However, in contrast to control nuclei, nuclei deficient in p97 fail to grow in size over time and do not replicate their chromosomal DNA. ssDNA replication in such extracts remains unaffected. Addition of the N-acetylglucosaminylated nuclear proteins of Xenopus or rat reverses these replication and growth defects. The possible role(s) of p97 in these nuclear functions is discussed.

Amino Acid Sequence↗

PubMed1995

Nuclear texture measurements in image cytometry.

DNA image cytometry is widely used in cytopathology as a means to obtain objective information concerning the diagnosis and prognosis of human cancer. Using specially designed devices, the high resolution spatial and photometric information is available in the images of a microscopic field. If quantitative DNA specific stains are used the chromatin distribution in the cell nuclei can be measured, which is one of the critical features for cytopathological analysis. In normal cells, changes in the chromatin appearance reflect changes in the activation patterns of genes. In tumors, dramatic changes in the nuclear chromatin appearance are common and have been associated with the progression of the disease. Features describing the chromatin distribution pattern are referred to as texture features. Nuclear texture features are sensitive to the differences between the various descriptive classes of chromatin patterns. In this paper we discuss the main categories of nuclear texture measurements. Texture features can be roughly divided into the following categories: 1) descriptive statistics of chromatin distribution; 2) discrete texture features; 3) range extreme; 4) markovian; 5) run length and 6) fractal texture features. Representative features of each of the above categories are discussed together with mathematical formulas, simple figures for explanation as well as images of typical cells which differ significantly in some texture features. Key references are also provided.

Cell Nucleus↗

PubMed1991

The target DNA sequence for resolution of poxvirus replicative intermediates is an active late promoter.

The linear double-stranded genomes of poxviruses such as Shope fibroma virus (SFV) replicate autonomously within the cytoplasm of infected cells, and it is believed that all of the replication functions are virally encoded. During DNA replication the incompletely base-paired terminal hairpin loops of the viral genome transiently exist in the form of inverted repeat replicative intermediates. These inverted repeat structures form the target for telomere resolution events that include sequence-specific cleavage and directed strand exchange to form the hairpin termini of progeny virus genomes. The terminal sequence domain which forms the telomere resolution target (TRT) shares considerable sequence similarity with viral late promoters. In this study we demonstrate that the TRT of SFV is capable of functioning as a strong viral promoter late in infection. A spectrum of TRT mutations affects telomere resolution and late transcription in a strictly concordant fashion, suggesting that the two activities may be inextricably linked. Further support for this concept comes from the demonstration that a late SFV promoter sequence designated cryptic TRT, which differs substantially from the native TRT in terms of sequence, can support telomere resolution when placed in the correct spatial context. The proposed model for telomere resolution invokes directed unwinding of the TRT double helix by a transcription initiation complex and processing of the resulting secondary structure by viral late-gene products.

Animals↗

PubMed1990

Tumorigenic poxviruses: characterization of the expression of an epidermal growth factor related gene in Shope fibroma virus.

The transcription and translation of an epidermal growth factor (EGF) related gene in the Leporipoxvirus Shope fibroma virus (SFV), termed the Shope fibroma growth factor (SFGF), have been characterized. Three early RNA transcripts complimentary to an anti-SFGF oligonucleotide were detected by Northern blot analysis, while no late transcripts were expressed. The activity of the SFGF early promoter was measured using a transient gene expression assay in SFV-infected cells using the bacterial choloramphenicol acetyltransferase as a reporter gene. Deletion analysis showed that the functional SFGF promoter domain is an AT-rich sequence contained within 30 bp of the major transcriptional initiation site as is typical of early poxvirus promoters. An intracellular form of the SFGF gene product was immunoprecipitated from infected lysates using rabbit antisera raised against a synthetic SFGF (amino acids 26-80). A 16-kDa product was detected, while in cells infected in the presence of tunicamycin, the immunoprecipitated product had a mobility on SDS-polyacrylamide gels of approximately 6 kDa, indicating that the SFGF gene product is extensively post-transcriptionally modified. The intracellular 16-kDa form can be pulse-chased to a 14-kDa form but the secreted form of SFGF could not be detected in the medium using this anti-peptide antiserum.

Base Sequence↗

PubMed1989

Tumorigenic poxviruses: characterization of an early promoter from Shope fibroma virus.

A strong early promoter from the T1 open reading frame (ORF) within the terminal inverted repeat (TIR) of Shope fibroma virus (SFV) has been isolated and characterized. Promoter activity was determined by a transient gene expression assay in poxvirus-infected cells using the bacterial chloramphenicol acetyltransferase as a reporter gene. The sequences which constitute the boundaries of the promoter element were determined by 5' and 3' deletion analysis. The functional SFV T1 promoter domain comprises about 28 bp and includes, in addition to the transcriptional initiation site, a stretch of eight continuous A residues from position -18 to -11 which is critical for promoter function. Both the SFV T1 promoter and the vaccinia 7.5-kDa early/late promoter are active in the transient expression assay when the cells are infected with either the leporipoxvirus SFV or the orthopoxvirus vaccinia. To look more closely at the conservation of promoter function between poxvirus genera, a recombinant vaccinia virus containing the CAT gene driven by the SFV T1 promoter and a recombinant SFV containing the CAT gene driven by the vaccinia 7.5-kDa early/late promoters was constructed. The SFV T1 promoter behaves as an early promoter in the vaccinia genome, and both the T1 and the 7.5-kDa early/late promoters use transcriptional initiation sites in their heterologous genomic environment that are identical to the ones used in the native viral genome. The results from this work indicate that despite the relative lack of absolute sequence conservation, the transcriptional machinery, at least with respect to temporal regulation of early promoters and the position of transcript initiation, is conserved between these two poxvirus genera.

DNA Mutational Analysis↗

PubMed1987

Tumorigenic poxviruses: transcriptional mapping of the terminal inverted repeats of Shope fibroma virus.

A composite transcriptional map for the entire 12.4-kb terminal inverted repeat (TIR) region of the Shope fibroma virus (SFV) genome has been determined. Northern blotting and S1-nuclease mapping were used to determine the regions which are transcribed, their temporal relationships, as well as the transcriptional initiation sites. Sequences representing the entire TIR are transcribed into poly(A)+ mRNA at both early and late times in the infection. Fifteen transcriptional initiation sites were mapped, 12 within the TIRs and 3 within the unique sequences close to the junction between the right TIR and the unique internal sequences. Ten of the 12 transcriptional initiation sites within the TIR and 2 of the 3 sites outside the right TIR correspond to the 5'-ends of the major open reading frames (ORFs) T1 to T9 plus the SFV growth factor gene. The 3 other initiation sites map within ORFs but near potential start codons for shorter polypeptides. All the expressed ORFs are tandemly arranged and transcribed toward the hairpin terminus. At early times during SFV infection of cultured rabbit cells, transcription of each ORF gives rise to a transcript of distinct size, while at late times termination of transcription is imprecise and substantial read-through into downstream sequences occurs. These results are discussed in light of recent observations on the related recombinant leporipoxvirus, malignant rabbit fibroma virus, which suggest that one or more gene products from this region of the SFV genome are implicated in viral tumorigenicity.

Base Sequence↗

PubMed1984

Tumorigenic poxviruses: construction of the composite physical map of the Shope fibroma virus genome.

The sites for the restriction enzymes BamHI, Bg/I, HindIII, PstI, PvuII, and SstI on the linear DNA genome of Shope fibroma virus, a tumorigenic poxvirus of rabbits, have been determined by digestions of the cloned BamHI and HindIII restriction fragments and by hybridization of 32P-labeled cloned fragments to Southern blots of Shope fibroma virus DNA cleaved partially or completely with the various enzymes. The linear genome is shown to be 160 kilobases in length and to possess terminal inverted repeat sequences of between 12.2 and 12.5 kilobases extending inwards from the cross-linked DNA telomeres. The fine map of the Shope fibroma virus terminal inverted repeats has been constructed and shown to be distinctly different from that of members of the orthopoxvirus group, such as vaccinia, by the absence of detectable tandemly repeated sequences near the termini and by the lack of detectable sequence homology with vaccinia termini.

Animals↗

PubMed1983

Physical characterization and molecular cloning of the Shope fibroma virus DNA genome.

DNA from several independent strains of Shope fibroma virus, a tumorogenic leporipoxvirus of rabbits, was isolated and analyzed by restriction endonuclease digestion and Southern blotting. The restriction profiles indicated a high degree of sequence conservation among the isolates but blotting under standard stringencies revealed no detectable cross homology with a member of the orthopoxvirus group, vaccinia. The genome of the fibroma virus was calculated to be in excess of 160 kilobases and shown to possess two features analogous to the orthopoxvirus group: (1) the terminal restriction fragments possess covalently closed hairpin structures; and (2) the terminal sequences are present as inverted repeats of greater than 10 kilobases. The terminal 3.6 kilobase BamHI restriction fragment was cloned in pBR322 after removal of the hairpin structure with mung bean single strand-specific endonuclease and addition of BamHI linkers. SFV sequences within this terminal region were shown, using 32P SFV cloned terminal probe, to have none of the sequence heterogeneity characteristic of vaccinia DNA termini. The remaining 20 internal SFV BamHI restriction fragments were propagated in bacterial plasmids either as intact fragments, or after secondary digestion with HindIII, and together constitute the complete cloned SFV sequence library.

Base Sequence↗

PubMedDate not supplied

Fluorescent location of abnormal cells in cell smears obtained from the lungs of patients with lung cancer.

A bronchoscope fitted with a brush was used to collect cells from patients with lung tumours. The cells were either made into smears or deposited as monolayers on microscope slides. Using a fluorescent probe for the cell surface enzyme, referred to as guanidinobenzoatase, abnormal cells were readily distinguished from normal cells which lacked this enzyme.

Aminacrine↗

PubMedDate not supplied

Neonatal tetanus.

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Female↗