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Biomedical subjects

D Rhodes

Publications and source records attributed to D Rhodes.

At least 19 recordsLinked to original sources
PubMed1992

Adjacent zinc-finger motifs in multiple zinc-finger peptides from SWI5 form structurally independent, flexibly linked domains.

Peptides containing either one, two or three of the three zinc-finger motifs from the yeast transcription factor SWI5 have been prepared by expression in Escherichia coli. The DNA binding characteristics of these peptides were investigated, and a two-dimensional nuclear magnetic resonance (n.m.r.) study undertaken to establish the three-dimensional structures of the two-finger peptide. The peptide containing fingers 1 and 2 binds sequence specifically to two thirds of the DNA binding site recognized either by intact SWI5 or by the isolated three-finger peptide, and hence has the correct tertiary fold for DNA recognition. These results also establish the polarity of DNA binding, since the N-terminal two fingers of SWI5 bind to the 5' end of the DNA binding site. Mild proteolysis of the three-finger peptide using trypsin results in a small number of discrete products, which is consistent with the presence of three structured mini-domains. Nearly complete n.m.r. signal assignments were obtained for two peptides containing finger 2 alone or fingers 1 + 2. Comparison of two-dimensional spectra of these peptides and others clearly shows that the NOE enhancements and chemical shifts characteristic of each finger are quite insensitive to the presence or absence of neighbouring fingers. This clearly indicates that adjacent zinc-finger domains are structurally independent in these peptides from SWI5. However, there must be some steric limitations on the possible relative orientations of the fingers, and to establish limits for these a set of structures for the peptide containing fingers 1 + 2 was calculated using the YASAP simulated annealing protocol in conjunction with n.m.r.-based constraints. A more detailed description of the three-dimensional structures of finger 1 and finger 2, and their relationship to other previously determined structures of single zinc-fingers, is given in the accompanying paper.

Amino Acid Sequence↗
PubMed1992

A new approach to the analysis of DNase I footprinting data and its application to the TFIIIA/5S DNA complex.

We have re-examined DNase I footprinting data for the binding of transcription factor IIIA (TFIIIA) to the 5S RNA gene, taking into account the protein-DNA contacts observed in the crystal structure of the DNase I/DNA complex (1, 2). This structure was not available when many of the original footprinting experiments on the TFIIIA/DNA complex were performed. In this way the pattern of DNase I cleavage can be interpreted to map out with greater precision the regions on the 5S DNA occupied by TFIIIA. Then, assuming the binding site for a zinc-finger may be the same as that found in the structure of the zinc-finger protein Zif268/DNA complex (3), and taking into account footprinting data for truncated forms of TFIIIA, the TFIIIA zinc-fingers were fitted within the permitted regions. On the basis of this, an alignment of the zinc-fingers of TFIIIA with its DNA binding site is proposed, which combines features of earlier models (4).

Animals↗
PubMed1992

Sequence-specific DNA binding by a two zinc-finger peptide from the Drosophila melanogaster Tramtrack protein.

We show that the DNA-binding domain of the Drosophila melanogaster regulatory protein Tramtrack consists of a 66 amino acid sequence containing two zinc-finger motifs and a short sequence N-terminal to the first finger motif. This short N-terminal sequence is essential for DNA binding and we suggest it is involved in maintaining the three-dimensional structure of the first finger domain, as has been seen in the nuclear magnetic resonance structure of one of the zinc-finger domains of the yeast transcription factor SW15. The characterization of the DNA-binding activity of this 66 residue peptide (delta 911zf) shows that it binds in a sequence-specific manner, as a monomer, to a natural target site with an apparent KD approximately 4 x 10(-7) M. The shortest delta 911zf binding site, which retains full affinity, consists of an 11 base-pair sequence with a one nucleotide overhang at each 5' end. DNase I, hydroxyl radical and methylation protection footprinting studies show that, in common with other zinc-finger proteins, delta 911zf binds in the major groove of DNA. The data presented are consistent with the zinc-fingers of Tramtrack contacting both strands of the DNA, and thus the binding differs in detail to that observed in the crystal structure of the three zinc-fingers of Zif268 complexed to their target DNA.

Amino Acid Sequence↗
PubMed1991

Detection of hereditary haemochromatosis in an HLA-identical pedigree showing discordance between HLA class I genes and the disease locus.

Hereditary haemochromatosis is a recessive disease in which primary hepatocellular carcinoma, complicating cirrhosis, is responsible for about one-third of deaths in affected homozygotes. We describe a unique HLA haplo-identical pedigree showing parent-to-offspring transmission of hereditary haemochromatosis in whom HLA typing studies, including class I and class II allogenotype analysis, were of no benefit in identifying affected homozygotes. However, affected siblings in the pre-cirrhotic stage of haemochromatosis, with apparent discordance between the haemochromatosis allele and class I loci on chromosome 6, were detected by undertaking a family study, using analysis of serum parameters of iron status in combination with magnetic resonance imaging (MRI). This pedigree emphasises the critical importance of genetic and non-invasive methods for the identification of asymptomatic homozygotes before cirrhosis develops.

Alleles↗
PubMed1991

Patients with HIV infection have a reduced proportion of lymphocytes expressing the IL2 receptor p55 chain (TAC, CD25).

Using a high-sensitivity immunofluorescence procedure, a proportion of blood lymphocytes can be shown to express the p55 chain of the IL-2 receptor (CD25, TAC) without in vitro stimulation. In this study, we measured the proportion of peripheral blood lymphocytes expressing CD25 in patients with a diagnosis of HIV infection and compared the results with cells from controls. The mean value for HIV-positive samples was 15%, while controls gave a mean value of 31%. The difference between the groups was highly significant (P less than 0.001, Mann-Whitney U test). When expression of CD25 was examined separately in CD4-positive and CD8-positive T cells and in B cells, there was a wider spread of values in the HIV group compared with controls, but no systematic difference. In the patient group studied (110 samples from 53 patients) there was a weak correlation between CD25 and CD4 expression (correlation coefficient r = 0.21, P less than 0.02), but there were patients with low CD25 and high CD4 as well as patients with high CD25 and low CD4, suggesting that CD25 can vary independently of changes in CD4 lymphocytes. Although the majority of very low values of CD25 (less than 10%) were found in patients with stage IV disease, such low values were also common in Stage II disease.

Adult↗
PubMed1991

Beyond zinc fingers: steroid hormone receptors have a novel structural motif for DNA recognition.

The highly conserved DNA-binding domain of the steroid hormone receptors contains two 'zinc finger'-like sequence motifs. The three-dimensional structure in solution has been determined using two-dimensional 1H nuclear magnetic resonance (NMR) spectroscopy and shows that the two 'zinc finger'-like motifs fold to form a single structural domain. The combination of this structural information and mutagenesis data reveals how this family of transcriptional regulators bind to DNA.

Amino Acid Sequence↗
PubMed1990

Solution structure of the DNA-binding domain of the oestrogen receptor.

Steroid hormone receptors control gene expression through binding, as dimers, to short palindromic response elements located upstream of the genes they regulate. An independent domain of approximately 70 amino acids directs this sequence-specific DNA binding and is highly conserved between different receptor proteins and related transcription factors. This domain contains two zinc-binding Cys2-Cys2 sequence motifs, which loosely resemble the 'zinc-finger' motifs of TFIIIA. Here we describe the structure of the DNA-binding domain from the oestrogen receptor, as determined by two-dimensional 1H NMR techniques. The two 'zinc-finger'-like motifs fold to form a single structural domain and are thus distinct from the independently folded units of the TFIIIA-type zinc fingers. The structure consists of two helices perpendicular to each other. A zinc ion, coordinated by four conserved cysteines, holds the base of a loop at the N terminus of each helix. This novel structural domain seems to be a general structure for protein-DNA recognition.

Amino Acid Sequence↗
PubMed1990

Effects of ethanol on reinforced variations and repetitions by rats under a multiple schedule.

Response sequences emitted by five Long-Evans rats were reinforced under a two-component multiple schedule. In the REPEAT component, food pellets were contingent upon completion of a left-left-right-right (LLRR) sequence on two levers. In the VARY component, pellets were contingent upon variable sequences (i.e., a sequence was reinforced only if it differed from each of the previous five sequences). The rats learned to emit LLRR sequences in the REPEAT component and variable sequences in VARY. Intraperitoneal injections of ethanol (1.25, 1.75, and 2.25 g/kg) significantly increased sequence variability in REPEAT, thereby lowering reinforcement probability, but had little effect on sequence variability in the VARY component. These results extend previous findings that alcohol impairs the performance of reinforced repetitions but not of reinforced variations in response sequences.

Animals↗
PubMed1989

A novel method for the purification of the Xenopus transcription factor IIIA.

We have developed a quick and simple purification method that yields large quantities of the Xenopus zinc finger protein transcription factor IIIA (TFIIIA). The protein is purified in the form of the 7S storage particle (TFIIIA/5S RNA complex) found in the ovaries of immature Xenopus. Our method yields 0.5 to 1 mg of pure 7S particle per ovary. It involves a high speed centrifugation step, fractionation on a gel filtration column and a precipitation step using calcium chloride. TFIIIA purified using this protocol retains full DNA binding activity and has the expected zinc ion content.

Animals↗
PubMed1989

The DNA binding site of the Xenopus transcription factor IIIA has a non-B-form structure.

On the basis of nuclease digestion studies we proposed that the DNA binding site of transcription factor IIIA (TFIIIA) may have an overall structure with A-type rather than B-type characteristics. This proposal was substantiated by the crystal structure of a part of the TFIIIA binding site. Recently, however, it has been reported that the binding site for TFIIIA is B-form in solution, thus implying that the conformation present in crystals is not the structure in solution. We have carried out a study using comparative circular dichroism (CD) spectroscopy of a number of double stranded deoxyoligonucleotides of different sequence, and known crystal structure. The correlation we have found between CD characteristics and certain structural parameters indicates that the solution and crystal structures of the TFIIIA binding site are closely related. This structure may be classed as an intermediate type, between A-form and B-form DNA.

Animals↗
PubMed1988

Zinc-finger motifs expressed in E. coli and folded in vitro direct specific binding to DNA.

The short sequence motif named 'zinc finger', first recognized repeated in tandem in the Xenopus transcription factor IIIA (TFIIIA), is also found in the yeast transcriptional activator SWI5 (ref. 3) and many other regulator proteins. Embedded in the 709-amino-acid polypeptide chain of SWI5 are three tandemly repeated zinc-finger motifs. Because the zinc fingers of TFIIIA are known to bind to DNA, it is probable that in the case of SWI5 these finger motifs also play an important, but not necessarily exclusive, role in the sequence-specific binding of the protein to DNA. To test this prediction we have expressed the 89-amino-acid sequence of the domain containing the three zinc fingers of SWI5 in Escherichia coli as a cleavable fusion protein, purified under denaturing conditions and folded in vitro. This experimental approach allows us to study directly both the metal requirement and DNA-binding properties of the isolated polypeptide. We find that zinc is required for specific DNA recognition and, most significantly, DNaseI protection studies show that the isolated three-fingered domain is sufficient for sequence-specific binding to DNA.

Amino Acid Sequence↗
PubMed1986

An underlying repeat in some transcriptional control sequences corresponding to half a double helical turn of DNA.

Transcription factor IIIA, which binds to the internal control region of the Xenopus 5S RNA gene has a novel structure consisting of nine tandemly repeated structural units. It was proposed by us that each unit interacts with about 5 bp of DNA. We show here that there is a periodicity on this scale in the DNA sequence and, by fine scale probing with nucleases, a corresponding structural repeat. Similar sequence periodicities are found in the internal control regions of other genes transcribed by RNA polymerase III, and also in the SV40 promoter and a monkey gene region to which the transcription factor Sp1 binds. We propose that transcription factor IIIA is the type of a novel class of transcription factors offering combinatorial possibilities for the specific recognition of DNA.

Animals↗