Large-scale immunomagnetic separation system for the removal of tumor cells from bone marrow.
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Animals↗
Biomedical subjects
T Moss
Publications and source records attributed to T Moss.
The putative promoter of a Xenopus laevis ribosomal gene is reduplicated.
With the aid of a novel poly-dA tailing-partial restriction technique and S1-protection mapping, the 5' terminal coding sequence for the 40S precursor ribosomal RNA of Xenopus laevis has been exactly identified. Since the promoter sequence for the 40S RNA should lie close to its 5' terminal coding sequence, we are able to conclude that the "Bam-Island" sequence reduplication (1) almost certainly represents a promoter reduplication.
Animals↗
Sequence organization of the spacer DNA in a ribosomal gene unit of Xenopus laevis.
A detailed restriction map was constructed for a cloned Xenopus laevis rDNA fragment containing the nontranscribed spacer (NTS) and external transcribed spacer (ETS) together with a portion of both the 18S and 28S rRNA genes. The NTS was found to contain at least three distinct repetitious areas. Region 1 has a repeating unit of approximately 100 bp. The primary structure of this unit has been determined by DNA sequencing. Region 2 is very similar in organization to region 3, and both have an alternating 81/60 bp arrangement as revealed by restriction with Alu I and DNA sequencing. It can be shown that the 81 and 60 bp canons are virtually identical to one another excepting a deletion/insertion of a 21 bp segment. Region 3 differs from region 2 in having sites for Sma I with its 81 bp units. Between these repeated DNA sequences there are two identical, nonrepetitive DNA sequences, each of which is centered around a Bam Hl site. Most of the ETS has been sequenced. It was found to be nonrepetitive and extremely rich in Cs. Close to the 5' end of the 18S coding sequence there is a DNA stretch very rich in purines. About 2.25 kb upstream from the Eco Rl restriction site bisecting the 18S structural gene there is a unique sequence which may be homologous to the 5' end of the 40S precursor RNA. Present evidence suggests that the boundaries between NTS and ETS occur farther downstream than was suggested by electron microscopic data. Sequencing has revealed that the spacer DNA of X. laevis contains different kinds of simple DNA sequences, but no evidence has been found that spacer DNA once arose by saltation of a 15 bp segment. The most surprising finding was that the spacer sequences around the Bam restriction sites (the Bam islands) show high homology with a sequence near the NTS/ETS interface. From the restriction and sequencing analyses it can be deduced that in recent evolutionary times the DNA sequences near the 5' end of the ribosomal transcription unit were reduplicated twice and displaced into spacer by saltation of an intervening short DNA sequence (the 60/81 bp canons). Possible implications of these evolutionary events for spacer functions are consisdered. The sequencing has also provided a molecular basis for a whole range of conclusions arrived at previously by indirect approaches, and these are discussed.
Animals↗
High-resolution proton-magnetic-resonance studies of chromatin core particles.
The binding of histones in chromatin core particles and in core particles depleted of histones H2A and H2B has been studied by high-resolution proton nuclear magnetic resonance (NMR) at 270 MHZ. At low ionic strengths it is shown that histones H3 and H4 are bound in the core particle. Further, whereas the apolar regions of H2A and H2B are also bound to the core particle, the basic N-terminal and C-terminal regions are more mobile and give rise to sharp resonances in the NMR spectrum of the core particle. Between 0.3 and 0.6 M NaCl there is further release of basic regions of histones H3 and H4 from the complex. The dissociation of the core particle between 0.6 and 2.0 M NaCl is accompanied by the release of the structured apolar regions of the histones as evidenced by the appearance of a complex aromatic spectrum and perturbed upfield ring-current-shifted methyl resonances. Arginine residues are implicated in the binding between histones and DNA and 69% of these residues are found in the apolar regions of the histones. The interactions between histones and DNA in the core particle thus involves H3 and H4 and the apolar regions of H2A and H2B. It is suggested that these basic regions of H2A and H2B have binding sites outside the core particle.
Animals↗
Nucleosomes, histone interactions, and the role of histones H3 and H4.
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Animals↗
Sites of histone/histone interaction in the H3 - H4 complex.
Sites of interaction between histones H3 and h4 have been probed by investigating complex formation, firstly between histone H4 and three peptides cleaved by chemical means from histone H3 (residues 1-90 and 1-120 using cyanogen bromide and residues 42-135 using N-bromosuccinimide), secondly between histone H3 and two peptides cleaved from histone H4 (residues 1 - 84 using cyanogen bromide and residues 38-102 using chymotrypsin) and thirdly between the H4 peptide (residues 38-102) and the three H3 peptides (residues 1-90, 1-120 and 42-135). The criterion for complex formation is the appearance of characteristic perturbed resonances in the aromatic region of the 270 - MHZ proton resonance spectrum of the peptide mixture. It is concluded that loss of 37 N-terminal residues from histone H4 and 41 N-terminal residues from histone H3 does not prevent complex formation, whilst the loss of 18 C-terminal residues from H4 and 45 C-terminal residues from H3 does prevent it; that last 15 C-terminal residues of H3 are, however, not required for forming a complex. The regions important for complex formation are therefore defined as residues 42-120 in histone H3 and residues 38-102 in histone H4.
Amino Acids↗
Studies on the role and mode of operation of the very-lysine-rich histone H1 in eukaryote chromatin. The three structural regions of the histone H1 molecule.
Limited digestion with trypsin of both calf thymus H1 histone and the fragment 1--120 of the H1 molecule has resulted in the isolation of the fragment 35--120. This fragment assumes a globular structure under physiological conditions of pH and ionic strength. The variable N-terminal portion of the molecule, up to residue 34, is not required for the formation of the H1 globular structure. Proton nuclear magnetic resonance (NMR) and ultracentrifugation studies show that the H1 histone molecule consists of three distinct structural domains under structuring conditions: a random coil 'nose' consisting of 35 to 40 residues from the N-terminal end; a globular 'head' involving the next approximately 80 residues; and a random-coil 'tail' of the remainder of the molecule.
Amino Acids↗
A nucleosome-like structure containing DNA and the arginine-rich histones H3 and H4.
The low-angle X-ray diffraction pattern from fibres of reconstituted H3/H4/DNA complexes is very similar to that of chromatin and has well defined maxima at 10.6, 5.4, 3.4 and 2.6 nm. Staphyloccal nuclease digestion of reconstituted H3/H4/DNA yields DNA fragments of length 49, 69, 100, 128, 193 and 255 b.p. as principal components. Comparison of the relative amounts of DNA fragments shows that the larger components (100 and 128 b.p.) increase with respect to the smaller (49 and 69 b.p.) as the histone to DNA ratio increases. A structural unit containing intergral of 65 b.p. of DNA and tetrameric (H3/H4)2 is proposed such that longer DNA fragments result from multiples of this unit. The principal nucleo-protein particle resulting from nuclease digestion contains 128/139 b.p. of DNA and has electrophoretic mobility very close to that of 'core' nucleosome. It probably represents a dimer of the basic structural unit.
Animals↗
A pH-dependent interaction between histones H2A and H2B involving secondary and tertiary folding.
It has been shown by high-resolution proton magnetic resonance (PMR) spectroscopy and circular dichroism (CD) that an H2A/H2B histone complex exists after salt extraction of these histones from chromatin and that this complex can be fully renatured from both urea-denatured acid-extracted and from urea-denatured salt-extracted histones. The histone complex is shown to involve specific secondary and tertiary structure. Formation of this complex is observed to be critically dependent on pH, occurring at and above pH 5. It cannot be induced below pH 5 by increase in ionic strength. From CD spectra the H2A/H2B complex is shown to contain about 37% alpha helix but no beta structure, the latter being confirmed by infrared spectroscopy in the 6-mum region. The PMR spectra show that the structured region includes most of the aromatic residues of both histones, at least two histidine residues of H2B and probably histidines 31 and 82 of histone H2A. The secondary structure of histones H2A and H2B is predicted using the Chou and Fasman procedure and comparisons are made between the predictions for histones of different species. These results in conjunction with the experimental evidence lead to the conclusion that at least residues 31-95 of H2A and residues 37-114 of H2B, i.e. the more apolar regions of the molecules, are involved in the tertiary structure of the H2A/H2B complex.
Amino Acid Sequence↗
Physical studies on the H3/H4 histone tetramer.
High-resolution proton magnetic resonance spectroscopy (270 MHz), circular dichroism, and infrared spectroscopies and ultracentrifugation studies have been carried out on the salt-extracted (H3/H4)2 tetramer from calf thymus. The tetramer contains about 29% alpha helix and no beta structure. It is denatured in 6 M urea but can be renatured simply by dialysis to water. The proton spectrum shows a number of perturbed resonances which are not observed in the spectra of either H3 or H4 alone. The observation of these resonances demonstrates that the tetramer contains some elements of tertiary structure. The overall appearance of the spectrum however is close to that of a partially denatured protein. Sedimentation velocity studies show the tetramer to have a frictional ratio of 1.99 in 50 mM acetate/50 mM bisulfite and thus to be hydrodynamically quite different from a globular protein. Two possible structural models compatible with the data are discussed.
Amino Acids↗