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J R Wells

Publications and source records attributed to J R Wells.

At least 37 records · Page 2Linked to original sources
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↗
PubMed1987

Characterization of the six chicken histone H1 proteins and alignment with their respective genes.

Six histone H1 subtypes and histone H5, isolated from chicken erythrocyte nuclei, were visualized on acid/urea polyacrylamide gels. Four of the H1 subtypes have been purified to homogeneity by fast protein liquid chromatography on a strong cation exchange column. The other two subtypes were obtained as enriched fractions from the same fast protein liquid chromatography experiments. Because six chicken H1 genes have been completely sequenced (Coles, L.S., Robins, A. J., Madley, L.K., and Wells, J. R. E. (1987) J. Biol. Chem. 262, 9656-9663), it was possible to assign each of the six H1 proteins to a specific gene after amino acid sequence analysis of peptides derived from the subtypes.

Amino Acid Sequence↗
PubMed1987

Spacer alterations which increase the expression of porcine growth hormone in E. coli.

Full-length porcine growth hormone (PGH) cDNA clones were isolated from a porcine pituitary cDNA library. When the coding portion of the PGH gene was cloned into an E. coli expression vector downstream from the powerful trc promoter, high levels of mRNA, but no protein were detected. Mutation directed by an oligodeoxynucleotide primer altered 5'-non-coding sequences and raised the level of PGH produced from undetectable to 15% of the total cellular protein. Alteration of four codons infrequently used by E. coli in the 5'-end of the gene produced no further increases.

Animals↗
PubMed1987

Bony ossicular fixation using 2-cyano-butyl-acrylate adhesive.

We investigated the effects of 2-cyano-butyl-acrylate (Histoacryl) on the ossicles of 11 of 12 adult guinea pigs. A small amount of the adhesive, placed precisely between the incus and its bony fossa in each middle ear, fixed the ossicles. Light microscopic investigation of the ears of animals killed at 3, 6, 9, and 12 months after surgery showed a bony union of the ossicle to its surrounding niche in 11 of 12 operated-on ears. Negligible inflammatory reaction was seen in areas of the middle ear not in contact with the adhesive. These observations suggest that 2-cyano-butyl-acrylate may be used to achieve a durable bony union in ossiculoplastic surgery with minimal side effects in the middle ear.

Animals↗
PubMed1987

Isolation and characterization of the porcine growth hormone gene.

A cosmid clone containing the entire porcine growth hormone (PGH) gene has been isolated using a full-length PGH cDNA as the hybridization probe. The gene within the cosmid was subcloned into plasmids and completely sequenced. The coding, promoter, and both 5'- and 3'-noncoding sequences of the PGH gene were found to be highly conserved when compared to the previously sequenced genes coding for rat, human and bovine growth hormones, and also to the human placental lactogen gene. The high degree of conservation between the 5'- and 3'-noncoding regions of the genes from these different species indicates that growth hormone genes may be evolving by some unusual mechanism. The PGH gene was found to contain the unusual variant GC donor splice site.

Amino Acid Sequence↗
PubMed1987

H5 gene specific trans-activation by nuclear extracts from avian erythroid cells.

Nuclear extracts from chicken erythroid cells selectively stimulate transcription of the chicken histone H5 gene (and not of other chicken histone genes) after coinjection into frog oocytes. This effect is shown to involve an enhancerlike activity, and a region of the H5 gene sufficient to mediate trans-activation is defined.

Animals↗
PubMed1986

Chicken histone genes retain nuclear matrix association throughout the cell cycle.

The association between histone genes and the nuclear matrix (NM) during periods of high (S-phase) and low (non-S-phase) transcriptional activity has been investigated with synchronized cells from a chicken erythroid cell line (abbreviated ts34). By DNase I and restriction enzyme analysis, these studies reveal that both core and linker histone genes (represented by H2A and H1 genes respectively) are attached to the NM independent of their transcriptional activity during the cell-cycle. The tissue-specific histone gene H5, expressed constitutively, is nuclear matrix (NM)-associated in ts34 cells but is found in the supernatant (S/N) fractions of a non-erythroid T-cell line. Furthermore, we show that DNA sequences necessary for NM-attachment of the H5 gene lie within a 780 base pair region spanning part of the coding and 5' non-translated region. Of the three non-histone genes investigated, beta-actin sequences are expressed and are NM-attached, feather keratin genes are not expressed and predominate in the S/N, and beta-globin genes although not expressed in the ts34 cell line used were found in the NM fraction. In this case the association may be fortuitous or may reflect an early event prior to transcription of globin genes in differentiating erythroid cells. These results generally support the notion that actively transcribed genes are NM-attached, but that attachment per se is not synonymous with transcription.

Actins↗
PubMed1986

Mutagenesis of conserved 5' elements and transcription of a chicken H1 histone gene.

In addition to readily identifiable TATA and CAAT boxes, chicken H1 histone genes contain a highly conserved G-rich region at about minus 80 and an A-rich H1 histone gene-specific motif some 120 bases upstream from the H1 mRNA cap site. The level of transcription from wild-type, 'G-box' and 'A-box' H1 deletion mutant templates was tested in Xenopus oocytes and in HeLa cells. Removal of the H1 gene-specific motif had no effect on H1 gene transcription in either assay system, whereas deletion of the G-rich sequence decreased H1 mRNA levels by about ten-fold in oocytes and in HeLa cells. At least in heterologous systems, the H1-gene specific A-rich region does not appear to influence the level of H1 gene transcription.

Animals↗