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J R Wells

Publications and source records attributed to J R Wells.

At least 19 recordsLinked to original sources
PubMed1992

Sequence of a cDNA encoding chicken high-mobility-group protein-2.

There are several members of the high-mobility-group (HMG) of DNA-binding proteins, including HMG-1, HMG-2, HMG-14 and HMG-17 [Johns: The HMG Chromosomal Proteins. Academic Press, London, 1982]. We report here sequences encoding the chicken HMG-2 protein of 207 amino acids (aa). This assignment is made on the basis of available data which indicate 89% homology of the chicken aa sequence to porcine HMG-2. This compares with 78-81% homology to the HMG-1 proteins of rat, hamster, human, porcine, and bovine origin.

Amino Acid Sequence↗
PubMed1992

Production and characterization of recombinant insulin-like growth factor-I (IGF-I) and potent analogues of IGF-I, with Gly or Arg substituted for Glu3, following their expression in Escherichia coli as fusion proteins.

The development of an efficient expression system for insulin-like growth factor-I (IGF-I) in Escherichia coli as a fusion protein is described. The fusion protein consists of an N-terminal extension made up of the first 46 amino acids of methionyl porcine GH ([Met1]-pGH) followed by the dipeptide Val-Asn. The latter two residues provide a unique hydroxylamine-sensitive link between [Met1]-pGH(1-46) and the N-terminal Gly of IGF-I. Downstream processing of the fusion proteins involved isolation of inclusion bodies, cleavage at the Asn-Gly bond, refolding of the reduced IGF-I peptide and purification to homogeneity. This expression system was also used to produce two variants of IGF-I in which Glu3 was substituted by either Gly or Arg to give [Gly3]-IGF-I and [Arg3]-IGF-I respectively. Production of milligram quantities of IGF-I peptide was readily achieved. The purity of the IGF-I, [Gly3]-IGF-I and [Arg3]-IGF-I was established by high-performance liquid chromatography and N-terminal sequence analysis. [Gly3]-IGF-I and [Arg3]-IGF-I were more potent than IGF-I in biological assays measuring stimulation of protein synthesis and DNA synthesis or inhibition of protein breakdown in rat L6 myoblasts. Both analogues bound very poorly to bovine IGF-binding protein-2 and slightly less well than IGF-I to the type-1 receptor on rat L6 myoblasts. We conclude that reduced binding to IGF-binding proteins rather than increased receptor binding is the likely explanation for the greater biological potency of the analogues compared with IGF-I.

Amino Acid Sequence↗
PubMed1992

Novel recombinant fusion protein analogues of insulin-like growth factor (IGF)-I indicate the relative importance of IGF-binding protein and receptor binding for enhanced biological potency.

An efficient expression system in Escherichia coli for several biologically active insulin-like growth factor-I (IGF-I) fusion peptide analogues is described. These novel IGF-I fusion protein analogues have properties that make them very useful reagents in the investigation of IGF-I action. The analogues comprise an IGF-I sequence and the first 11 amino acids of methionyl porcine growth hormone (pGH) and include [Met1]-pGH(1-11)-Val-Asn-IGF-I, which contains the authentic IGF-I sequence, and two analogues, [Met1]-pGH(1-11)-Val-Asn-[Gly3]-IGF-I and [Met1]-pGH(1-11)-Val-Asn-[Arg3]-IGF-I, where Glu-3 in the human IGF-I sequence has been replaced by Gly or Arg respectively. The three peptides are referred to as Long IGF-I, Long [Gly3]-IGF-I or Long [Arg3]-IGF-I depending on the IGF-I sequence present. Production of the purified fusion peptides was aided by folding the reduced and denatured fusion peptide sequence under conditions that gave very high yields of biologically active product. Introduction of a hydrophobic N-terminal extension peptide appears to facilitate the correct folding of the IGF-I analogues compared with that obtained previously when folding normal-length IGFs. The biological activities of the IGF-I fusion peptides were compared with authentic IGF-I and the truncated analogue, des(1-3)IGF-I. In L6 rat myoblasts, all the analogues were more potent than authentic IGF-I in their abilities to stimulate protein and DNA synthesis and inhibit protein breakdown. In H35 hepatoma cells, where the IGFs act through the insulin receptor, the Long IGF-I analogues maintained a similar potency relative to IGF-I as was observed in the L6 myoblasts. The order of biological potency in cell lines secreting IGF-binding proteins (IGFBPs) into the medium was Long [Arg3]-IGF-I-des(1-3)IGF-I greater than Long [Gly3]-IGF-I greater than Long IGF-I greater than IGF-I. In chicken embryo fibroblasts, a cell line that does not secrete detectable IGFBPs into the medium, Long [Arg3]-IGF-I, was less potent than IGF-I. Investigation of receptor and IGFBP association by these analogues reinforced our previous findings that N-terminal analogues of IGF-I show increased biological potency due to changes in the degree of their IGFBP interactions.

Amino Acid Sequence↗
PubMed1991

Isolation and characterization of human thioredoxin-encoding genes.

Thioredoxin (Trx) has recently been demonstrated to be an essential component of the early pregnancy factor activity of pregnancy serum. Here, we report the structure and sequence of human Trx-encoding genes (Trx) by analysis of genomic clones. The Trx gene extends over 13 kb and consists of five exons encoding a 12-kDa protein. A 700-bp fragment upstream from the start codon functions as a promoter when inserted in front of a human growth hormone-encoding reporter gene in tissue-culture cells. This promoter region is very G + C rich and does not contain a classical TATA or CCAAT box, but has three consensus sequences for high-affinity Sp1 binding. Southern analysis demonstrated the presence of several Trx genes in the human genome. The number includes at least one inactive copy as shown by the isolation and sequencing of an inactive pseudogene.

Base Sequence↗
PubMed1991

Growth and protein production kinetics of a murine myeloma cell line transfected with the human growth hormone gene.

A model mammalian cell system for the production of recombinant proteins was investigated. Murine myeloma cells which had lost the ability to produce both heavy and light chain immunoglobulin molecules were transfected with a vector containing the immunoglobulin heavy chain promoter and enhancer elements linked to the human growth hormone gene. The growth kinetics of G32, a clonal isolate, were found to be similar to both the parent myeloma and hybridomas. However, production of hGH by G32 was growth associated, rather than as a secondary metabolite as is the case for hybridomas. In addition, G32 produced hGH at molar levels greater than most hybridomas.

Animals↗
PubMed1991

Identification of molecules involved in the 'early pregnancy factor' phenomenon.

An isolated preparation from ovine placental extracts which was active in the rosette inhibition assay mimicking the activity of the so-called 'early pregnancy factor' (EPF) has been shown to contain a 12 kDa polypeptide which could be partially resolved from low-molecular-weight active moieties. N-terminal amino acid sequence analysis of the polypeptide indicated that it was ovine thioredoxin, an identification confirmed by isolation and complete sequence analysis of the corresponding cDNA. The cDNA for human thioredoxin was expressed in Escherichia coli and the recombinant protein isolated and purified. Pure recombinant thioredoxin alone did not induce the expression of increased rosette inhibition titres (RITs) when tested in the rosette inhibition assay; but, when tested in combination with cell stimuli such as platelet-activating factor (PAF) or serum, it allowed the expression of increased RITs where none was achieved in its absence. Thioredoxin acted in the assay to reverse a refractory state normally induced by these stimuli, allowing lipoxygenase-dependent moieties also induced by the stimuli to exert their effects, resulting in the expression of increased RITs. Antibodies to recombinant thioredoxin removed from pregnancy sera the capacity to induce increased RITs, i.e. to express EPF activity, thus establishing a role for thioredoxin or thioredoxin-like proteins and associated molecules in the mechanisms which allow pregnancy sera to induce increased RITs. Based on a consideration of these and other results, a new model for the study of the EPF phenomenon is presented and discussed.

Amino Acid Sequence↗
PubMed1991

Expression, purification and characterization of secreted recombinant human insulin-like growth factor-I (IGF-I) and the potent variant des(1-3) IGF-I in Chinese hamster ovary cells.

Recombinant human insulin-like growth factor-I (hIGF-I) and a biologically potent variant lacking the N-terminal tripeptide (des(1-3)IGF-I) were produced from transfected Chinese hamster ovary cells. The constructs encoding the signal peptide, sequence of the mature peptide and a C-terminal extension peptide were expressed under the control of a Rous sarcoma virus promoter. Successfully transfected clones secreting correctly processed recombinant hIGF-I or des(1-3)IGF-I were selected by their secretion of IGF-I-like activity into the culture medium. The recombinant peptides were purified to homogeneity as assessed by high-performance liquid chromatography and N-terminal sequence analysis. The purified recombinant peptides exhibited biological potencies equivalent to authentic IGF-I and des(1-3)IGF-I respectively.

Amino Acid Sequence↗
PubMed1990

Crystallization of the globular domain of histone H5.

The globular domain of histone H1/H5 binds to the nucleosome and is crucial for the formation of chromatin higher order structure. We have expressed in Escherichia coli a gene that codes for the globular domain of H5. The protein produced in E. coli is functional in nucleosome binding assays. We have obtained crystals of the protein that diffract to beyond 2.5 A (1 A = 0.1 nm) resolution. The crystals are orthorhombic with unit cell dimensions of a = 80.1 A, b = 67.5 A and c = 38.0 A.

Crystallization↗
PubMed1989

Transcription from the intron-containing chicken histone H2A.F gene is not S-phase regulated.

The nucleotide sequence of an 8.2 kb BamHI fragment containing the entire chicken histone H2AF gene has been determined. Unlike the majority of histone genes, the coding region is interrupted by four intervening sequences. While sequencing the 8.2 kb BamHI fragment it was found that the promoter and first exon of an unidentified non-histone gene lies immediately downstream of the H2AF gene. Studies of H2AF gene transcription show that, unlike the major core and H1 histone genes, it is not coupled to DNA synthesis.

Amino Acid Sequence↗
PubMed1988

Conservation of histone H2A/H2B intergene regions: a role for the H2B specific element in divergent transcription.

The organization and function of potential regulatory elements associated with the promoters of chicken H2A and H2B genes pairs have been examined. The intergene regions of six dispersed and divergently-transcribed H2A/H2B gene pairs contain several extremely well conserved and spaced blocks of sequence homology. Adjacent coding regions are on average 342 base-pairs apart. Respective TATA boxes are separated by 180 base-pairs and within this confined region there are four CCAAT boxes and a previously identified 13 base-pair H2B-specific element (H2B-box) which has homology to the octamer motif present in a number of gene promoter/enhancer elements. Transcription of H2A and H2B genes from wild-type and mutant constructs was measured in transient assays by transfection into HeLa cells, and in permanently transformed clonal cell lines. In vitro separation of the two genes at a unique intergenic site significantly decreased transcription of each gene. This suggested that the H2A/H2B gene pairs contained overlapping promoters. Deletion or point mutagenesis of the H2B-specific element decreased the levels of H2B and the H2A transcripts indicating that this sequence is a common regulatory element of both genes in the divergent-pair configeration.

Animals↗
PubMed1988

A gene-specific promoter element is required for optimal expression of the histone H1 gene in S-phase.

An H1 gene-specific element (H1-box, 5'-AAACACA-3') modulates S-phase expression of the gene in vivo as judged by analysis of transcripts from histone genes transfected into HeLa cells. Deletion or base-substitution of the element causes a 15- to 30-fold decrease in steady-state H1 mRNA levels in randomly growing cells and eliminates cell cycle control of transcription in synchronized cells. Mutations within the H1-specific element which abolish S-phase control of transcription also eliminate binding of a sequence-specific nuclear factor capable of binding specifically to this region in vitro. Transfection of multiple copies of H1-box elements into cells drastically decreases H1 mRNA levels, mimicking the effect observed when the motif is rendered non-functional by deletion or substitution mutagenesis. In contrast, introduction of mutated H1 elements into cells has no detectable effect. Together, these results imply that an interaction between the H1-box and a sequence-specific trans-acting factor modulates transcriptional control of H1 genes in vivo.

Alpharetrovirus↗
PubMed1988

Maximal binding levels of an H1 histone gene-specific factor in S-phase correlate with maximal H1 gene transcription.

Levels of trans-acting factor (H1-SF) binding to the histone H1 gene-specific motif (5'-AAACACA-3' [L. S. Coles and J. R. E. Wells, Nucleic Acids Res. 13:585-594, 1985]) increase 12-fold from G1 to S-phase in synchronized cells and decrease again in G2 phase of the cell cycle. Since the H1 element is required for S-phase expression of H1 genes (S. Dalton and J. R. E. Wells, EMBO J. 7:49-56, 1988), it is likely that the increased levels of H1-SF binding component play an important role in S-phase regulation of H1 gene transcription.

Animals↗
PubMed1988

Functional and phenotypic characterization of human B lymphocyte subsets isolated by unit gravity sedimentation.

We report the use of unit gravity sedimentation with a CelSep apparatus to generate two volumetrically similar but functionally and phenotypically distinct subsets of human peripheral blood B cells. One subset, comprised of small B lymphocytes, underwent a significant size change in response to anti-mu, proliferated synergistically to low concentrations of anti-mu plus B cell growth factor (BCGF) or phorbol myristate acetate plus BCGF, and could be induced to produce immunoglobulin in response to pokeweed-mitogen-derived T-lymphocyte-replacing factors. These cells were primarily sIg+, B1+, B2+, and were virtually free of monocytes (less than 0.01%). Unlike these resting B lymphocytes, the large cells proliferated directly to BCGF, without displaying synergy with anti-mu. These cells displayed very little B2 (less than 7%), did not increase in volume in the presence of anti-mu, and made more immunoglobulin in response to TRF than the small resting B lymphocytes. However, neither population synthesized immunoglobulin spontaneously. This technique, which is highly reproducible, not equipment intensive, and produces high cell recovery (greater than 90%), allows for a precise analysis of the steps involved in the maturation of a resting B lymphocyte to an immunoglobulin-secreting cell.

B-Lymphocytes↗
PubMed1988

Introduction of a porcine growth hormone fusion gene into transgenic pigs promotes growth.

Six transgenic pigs have been produced by microinjecting a human metallothionein promoter/porcine growth hormone gene construct into the pronuclei of fertilized eggs which were transferred to synchronized recipient sows. The resulting transgenic animals contained between 0.5 and 15 copies of the gene construct per cell, and at least one of the animals expressed the introduced gene and grew at an increased rate compared to both transgenic and non-transgenic littermates. Some of the transgenic animals that did not appear to grow at increased rates were found to contain rearranged gene sequences. Two of the transgenic pigs have been shown to pass on the introduced genes to their offspring.

Animals↗
PubMed1987

Characterization of the chicken histone H1 gene complement. Generation of a complete set of vertebrate H1 protein sequences.

Sequence analysis of four chicken H1 histone genes described here completes the characterization of the full complement of six H1 genes in the chicken genome. Each of the six genes codes for a different H1 protein sequence, and these range in size from 217 to 224 amino acids. The proteins are distinct in sequence from the H1-related chicken H5 protein and appear to be analogous to the standard somatic mammalian H1 subtypes. The protein sequence data deduced from the genes represent the first complete set of vertebrate H1 protein sequences. Comparison of the chicken H1 gene noncoding sequences with each other and with H1 gene sequences from other organisms reveals conservation of an H1 gene-specific element, a G-rich element, and histone gene-specific 3' elements. Additional sequences are conserved between H1 genes of the chicken and other vertebrates. Comparisons also reveal variation in promoter and 3' elements between chicken genes that could play a role in the differential expression of H1 gene protein products.

Amino Acid Sequence↗