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C Crane-Robinson

Publications and source records attributed to C Crane-Robinson.

At least 55 records · Page 3Linked to original sources
PubMed1990

Thymosins: both nuclear and cytoplasmic proteins.

A simple procedure based on perchloric acid extraction has been developed for the preparation and purification of bovine prothymosin alpha and thymosins beta 4 and beta 9 in high yields. Spectroscopic observations show these proteins to be non-folding at neural pH. The cellular locations of human prothymosin alpha, rat parathymosin and calf thymosin beta 4, all so-called 'thymic hormones', have been studied by injection into the cytoplasm of Xenopus oocytes, followed by separate monitoring of nuclear and cytoplasmic concentrations. It is shown that human prothymosin alpha and rat parathymosin both migrate to the nucleus whilst thymosin beta 4 remains in the cytoplasm. The peptide (1-88) of calf prothymosin alpha is shown not to accumulate in the Xenopus nucleus, demonstrating that the C-terminal 21 residues, which include a KKQK sequence, are required for nuclear migration. The present data, in association with existing evidence of wide tissue distribution and the lack of signal peptides, indicate that these proteins do not behave as hormones in the usual sense of the word. It is suggested that thymosin beta 4 should be grouped separately from the pro- and parathymosins.

Amino Acid Sequence↗
PubMed1990

1H-n.m.r. studies of the isolated activation segment from pig procarboxypeptidase A.

The isolated activation segment (asA) from pig pancreatic procarboxypeptidase A was studied by 1H-n.m.r. spectroscopy over a wide range of solution conditions. Isolated asA shows many characteristics of compactly folded globular proteins, such as the observation of perturbed positions for resonances from methyl groups, alpha-carbon atoms, histidine residues and the tyrosine residue. The single tyrosine residue (Tyr-70) exhibits a very high pKa, and both histidine and tyrosine residues show slow chemical modification (deuteration and iodination). In contrast, asA shows rapid NH exchange. Analysis of the spectra by pH titration and nuclear Overhauser effects revealed several residue interactions. Quantitative analysis of deuterium and tritium exchange allowed the assignment of the histidine C-2-H resonances to their respective residues in the sequence. His-66, the closest to the sites of proteolytic attack in the proenzyme, is shown to be the most accessible to solvent in procarboxypeptidase A. It was also shown that asA is thermally very stable ['melting' temperature (Tm) 88 degrees C] and requires a high urea concentration for denaturation (6.25 M, at pH 7.5). Evidence is presented for some degree of conformational flexibility in the premelting range, a feature that could be ascribed to the preponderance of helical secondary structure and to the lack of disulphide bridges. The free solution structure of asA is probably unchanged when it binds to carboxypeptidase A.

Animals↗
PubMed1989

Prothymosin alpha is a nuclear protein.

The cellular location of the so-called 'thymic hormone' prothymosin alpha has been studied by microinjection into the cytoplasm of Xenopus oocytes, followed by separate monitoring of nuclear and cytoplasmic concentrations. It is shown that prothymosin alpha migrates to the nucleus at a rate comparable to that of histone H1. Prothymosin alpha cannot therefore be a hormone in the usual sense of the word.

Animals↗
PubMed1989

A "minimal epitope" anti-protein antibody that recognises a single modified amino acid.

Antibodies that recognise proteins bind to epitopes of varying size, but a grouping of the order of six amino acids, contiguous or not, is regarded as a typical number. By using as immunogen a highly abundant and universal eukaryotic nuclear protein (histone H4) modified in a manner not typical of secreted proteins (acetylation of lysine side chains), antiserum has been raised in rabbits having the single amino acid epsilon-N-acetyl lysine as the recognition epitope. The affinity-purified antibody should be useful for studying the functional role of this modification. The methodology has potential for raising antibodies to other types of post-translationally modified proteins.

Animals↗
PubMed1989

Binding of the globular domain of linker histones H5/H1 to the nucleosome: a hypothesis.

The amino acid sequence of the central globular domain of histone H1/H5 family members is highly homologous. Twenty-four such sequences have been compared to establish the conserved and variable residues. Fitting this to the tertiary structure of the H5 globular domain shows which of the conserved and variable residues are peripheral and which internal. Particular attention is paid to conserved basic residues on the surface, which we take to be DNA binding. Variable regions and conserved acidic residues are assumed not to be sites of contact with DNA. We conclude that one face of the domain, containing a cluster of basic residues, is the principal DNA binding site whilst two opposing faces, orthogonal to the principal site and also containing conserved basic residues, are subsidiary DNA binding sites. Since the DNA binding surface of the domain covers a full 180 degrees arc, we propose that it contacts a 'cage' of three DNA strands on the 2-fold axis of the chromatosome.

Amino Acid Sequence↗
PubMed1988

Footprinting of linker histones H5 and H1 on the nucleosome.

DNase I has been used to footprint the linker histones H5 and H1 on the nucleosome of chicken erythrocyte chromatin. Rate constants have been derived for digestion at the principal sites of attack on chromatosome length DNA (168 bp), located about 10 bp apart, and compared with those observed for linker histone-depleted chromatosomes. Complete protection was found for site S7 on the dyad axis and decreasing partial protection seen at symmetrically positioned sites on each side of S7. Strong, but not complete protection was noted at S14, the site corresponding to the end of the core particle, situated less than 1/4 of a turn away from the dyad. Uniform partial protection was observed for sites S2, S3, S4 and S10, S12 on the far side of the chromatosome. The simplest interpretation of these results is that the globular domain of H5/H1 is responsible for the protection at S7, whilst extended N- and C-domains give rise to the partial protection at sites away from the dyad axis.

Animals↗
PubMed1988

A direct link between core histone acetylation and transcriptionally active chromatin.

An antiserum raised against chemically acetylated histone H4 was found to recognize the epitope epsilon-N-acetyl lysine. Affinity-purified antibodies were used to fractionate oligo- and mononucleosomal chromatin fragments from the nuclei of 15-day chicken embryo erythrocytes. Antibody-bound chromatin was found to contain elevated levels of acetylated core histones. On probing with sequences of alpha D globin, an actively transcribed gene, the antibody-bound chromatin was 15- to 30-fold enriched relative to the input chromatin. Using ovalbumin sequences as a probe, no enrichment was observed. The results demonstrate directly that transcriptionally active genes carry acetylated core histones.

Acetylation↗
PubMed1987

The chromatin of sea urchin sperm.

Digestion of sea urchin sperm nuclei with micrococcal nuclease yields nucleosomal monomer fragments of 151 and 164 base pairs. Prior trypsin treatment of the sperm chromatin does not alter the size of these monomer DNA fragments despite the fact that the H1 histone is reduced to a limit globular peptide of about 83 residues. Heterologous reconstitution experiments show that this peptide is capable of protecting an extra 22 base pairs beyond the core particle in a chromatosome. Nuclease digestion of reconstitutes from DNA and sperm core histones yields a core monomer of about 141 base pairs. It is concluded that this sperm chromatin contains a chromatosome of 164 bp essentially similar to that observed in the more usual chromatins. Edman degradation of the H1 limit peptide shows its sequence to be closely analogous to the corresponding peptide of calf H1 and chicken H5. Circular dichroism studies of histone H1 from the sperm of three sea urchin species demonstrate the presence of trypsin-sensitive helical regions outside the globular domain that are absent in calf H1 and chicken H5.

Amino Acid Sequence↗
PubMed1987

Elevated levels of a specific class of nuclear phosphoproteins in cells transformed with v-ras and v-mos oncogenes and by cotransfection with c-myc and polyoma middle T genes.

Transformation of a rat thyroid epithelial cell line (FRTL5-C12) with Kirsten and Harvey murine sarcoma viruses (carrying the ras oncogenes) results in elevated levels of three perchloric acid-soluble nuclear phosphoproteins. These three proteins are also induced to high levels in the PC-C13 thyroid epithelial cell line when transformed by the myeloproliferative sarcoma virus (carrying the v-mos oncogene) and when transformed by transfection with the c-myc proto-oncogene followed by infection with the polyoma leukaemia virus (PyMuLV) carry the polyoma middle T antigen gene. Neither c-myc or PyMuLV alone induced high levels of the three nuclear proteins. Untransformed thyroid fibroblasts have high levels of two of the three proteins and can be transformed by PyMuLV alone resulting in the appearance of the third protein. Transformation with Harvey sarcoma virus also results in the induction of the third protein. The three phosphoproteins have been purified by h.p.l.c. and shown to be related to the HeLa protein HMGI already described. The results of these studies indicate that elevated levels of these HMGI-like proteins are associated with neoplastic transformation and/or with an undifferentiated phenotype.

Animals↗
PubMed1987

The structure of ubiquitinated histone H2B.

Ubiquitinated histone H2B (uH2B) has been purified from both calf and pig thymus by exclusion chromatography in 7 M urea. Digestion of uH2B with Staphylococcus aureus V8 protease yielded the peptide 114-125 containing the ubiquitin moiety. Further digestion of this peptide with trypsin removed the ubiquitin and three H2B residues from the N-terminus. Edman degradations of both peptides established that ubiquitin is attached to the epsilon-amino group of lysine 120 in both calf and pig uH2B by an iso-peptide bond to the C-terminal glycine 76 of ubiquitin.

Amino Acid Sequence↗
PubMed1987

Histone and nonhistone proteins from normal and virus-transformed rat thyroid epithelial cells.

The histone and nonhistone nuclear proteins extracted by different methods from nuclei of normal and virus-transformed rat thyroid epithelial cells (FRTL5 cell line) were studied by polyacrylamide gel electrophoresis in acetic acid/urea and in SDS and by autoradiography. The results have shown that some of these proteins have an higher level of phosphorylation in virus-transformed than in normal cells; moreover, an higher amount of three proteins (C, D, and E), which in normal cells are not detectable at least as Coomassie staining, was found. These proteins, extractable with perchloric acid, are suggested to belong to the High Mobility Group (HMG proteins) and to play some regulatory role.

Animals↗
PubMed1986

Roles of H1 domains in determining higher order chromatin structure and H1 location.

Peptides derived from calf thymus H1 and rat liver H1, comprising only the globular and COOH-terminal domains of the intact molecule and therefore lacking NH2-terminal domains, have been shown by reconstitution to be as effective as the complete H1 molecule in inducing higher-order-chromatin structure. As the globular domain of H1 alone cannot induce chromatin folding, our results demonstrate that this function is primarily controlled by the COOH-terminal domain of the molecule. Surprisingly, these peptides do not locate correctly with respect to the nucleosome. This is demonstrated by their failure to confer upon reconstitutes the ability to protect DNA fragments of chromatosome length when digested with micrococcal nuclease. The precise placement of the H1 molecule (globular domain) with respect to the nucleosome is shown to be influenced by the "tail" domains of both H1 and the core histones.

Animals↗
PubMed1985

The structure of sub-nucleosomal particles. The octameric (H3/H4)4--125-base-pair-DNA complex.

Chicken erythrocyte chromatin was depleted of histones H1, H5, H2A and H2B. The resulting (H3/H4)-containing chromatin was digested with micrococcal nuclease to yield monomer, dimer, trimer etc. units, irregularly spaced on the DNA, with even-number multimers being more prominent. Sucrose density gradient centrifugation separated monomers and dimers (7.7 S and 10.5 S). Sodium dodecyl sulphate gel electrophoresis and cross-linking indicated: the monomer contains 50-base-pair (bp), 60-bp and 70-bp DNA and the dimer 125-bp DNA; the monomer contains a tetramer and the dimer an octamer of H3 and H4. Partial association of monomer units to dimers inhibits structural studies of monomers. The internal structure of the dimer, i.e. and (H3/H4)4-125-bp-DNA particle, was studied using circular dichroism, thermal denaturation and nuclease digestion. Both micrococcal nuclease and DNase I digestion indicate that, unlike core particles, accessible sites occur in the centre of the particle and it is concluded that the (H3/H4)4-125-bp-DNA particle is not a 'pseudo-core particle' in which the 'extra' H3 and H4 replace H2A and H2B. It is proposed that the octamer particle is formed by the sliding together of two 'monomer' units, each containing the (H3/H4)2 tetramer and 70 bp of DNA. Excision of this dimer unit with micrococcal nuclease results in the loss of 10 readily digestible base pairs at each end, leaving 125 bp.

Animals↗
PubMed1985

Structure of subnucleosomal particles. Tetrameric (H3/H4)2 146 base pair DNA and hexameric (H3/H4)2(H2A/H2B)1 146 base pair DNA complexes.

The tetrameric (H3/H4)2 146 base pair (bp) DNA and hexameric (H3/H4)2(H2A/H2B)1 146 bp DNA subnucleosomal particles have been prepared by depletion of chicken erythrocyte core particles using 3 or 4 M urea, 250 mM sodium chloride, and a cation-exchange resin. The particles have been characterized by cross-linking and sedimentation equilibrium. The structures of the particles, particularly the tetrameric, have been studied by sedimentation velocity, low-angle neutron scattering, circular dichroism, optical melting, and nuclease digestion with DNase I, micrococcal nuclease, and exonuclease III. It is concluded that since the radius of gyration of the DNA in the tetramer particle and its maximum dimension are very close to those of the core particle, no expansion occurs on removal of all the H2A and H2B. Nuclease digestion results indicate that histones H3/H4 in the tetramer particle protect a total of 70 bp of DNA that are centrally located within the 146 bp. Within the 70 bp DNA length, the two terminal regions of 10 bp are, however, not strongly protected from digestion. The optical melting profile of both particles can be resolved into three components and is consistent with the model of histone protection of DNA proposed from nuclease digestion. The structure proposed for the tetrameric histone complex bound to DNA is that of a compact particle containing 1.75 superhelical turns of DNA, in which the H3 and H4 histone location is the same as found for the core particle in chromatin by histone/DNA cross-linking [Shick, V. V., Belyavsky, A. V., Bavykin, S. G., & Mirzabekov, A. D. (1980) J. Mol. Biol. 139, 491-517]. Optical melting of the hexamer particle shows that each (H2A/H2B)1 dimer of the core particle protects about 22 base pairs of DNA.

Animals↗
PubMed1985

Changes in nuclear proteins on transformation of rat epithelial thyroid cells by a murine sarcoma retrovirus.

Two-dimensional electrophoresis has been used to document changes in nuclear proteins following viral transformation of an epithelial cell line exhibiting differentiation markers. After transformation, these markers are lost, and the cells become tumorigenic and capable of growth in soft agar. A sharp rise in the phosphorylation of histones H1, H2A, and ubiquitinated H2A is seen on transformation, together with the appearance of three phosphorylated proteins that are extractable by perchloric acid and appear related to high mobility group Protein 14, a constituent of active chromatin. Since comparison is made between normal and transformed cells that are each grown to confluence and since there is little difference between their observed growth rates, the changes seen represent intrinsic differences between the cell lines and are thus a direct reflection of the process of transformation.

Animals↗
PubMed1984

Conformation and domain structure of the non-histone chromosomal proteins HMG 1 and 2. Domain interactions.

The sequence of the 224 residues of HMG 1 suggests it consists of three domains. We have previously proposed [Cary et al. (1980 Eur. J. Biochem. 131, 367-374] that the A and B domains can fold autonomously and that there is also a small N domain. Several proteases are now found to cut at the end of the B domain (at or close to residue 184). It is shown that the A + B-domain fragment also folds and probably contains all the helix of intact HMG 1. The stability of the B domain is enhanced by the presence of the A domain. The acidic C domain undergoes a coil----helix transition on lowering the pH. Several peptides have been prepared by cleavage at tryptophan. Peptide 57--C-terminus contains complete B and C domains but does not fold. In the absence of the A domain the C domain is thus able to destabilise the B domain. It is concluded that the stability of the B domain in HMG 1 is due to interaction with the A domain and the C domain has a separate function from the other domains.

Amino Acids↗
PubMed1984

Histone modification in early and late Drosophila embryos.

Levels of histone acetylation and phosphorylation have been contrasted in two developmental states of Drosophila melanogaster. The 0-2 h nuclei of the syncitial blastula are characterized by rapid mitoses and DNA replication, but there is very little transcription. In the 18 h embryo there is considerable transcription and the mitotic rate is much slower. It has been found that (1) histone H1 from 2h nuclei is not highly phosphorylated. This observation is not in accord with the view that H1 hyperphosphorylation is essential to mitosis, but is compatible with the hypothesis that H1 phosphorylation in Drosophila species is related to heterochromatization. (2) Histone H4 from 2 h embryos shows high levels of the diacetyl form (H4-Ac2), which is principally outside the nucleus. This accords with the hypothesis that H4-Ac2 is the form in which H4 is deposited on to newly replicated DNA and shows that H4 acetylation is linked not only to transcription. (3) Histone H3 acetylation is similar in 2h and in 18h embryos. As with H4, this acetylation probably correlates with chromatin assembly and is not transcription-related. (4) Histone H2B carries no modification in 2h or in 18h embryos, and H2A shows a single modification in 2h embryos and two in 18 h embryos. H2B modification is thus not essential either in mitosis or replication, whereas H2A modification is important in one or both processes. (5) The nucleosomal protein D2 is equally present in 2h and 18 h embryos.

Acetylation↗