Search PubMed⌕ Search

Biomedical subjects

D W Burt

Publications and source records attributed to D W Burt.

At least 55 records · Page 3Linked to original sources
PubMed1998

Characterization of whole genome radiation hybrid mapping resources for non-mammalian vertebrates.

Radiation hybrid panels are already available for genome mapping in human and mouse. In this study we have used two model organisms (chicken and zebrafish) to show that hybrid panels that contain a full complement of the donor genome can be generated by fusion to hamster cells. The quality of the resulting hybrids has been assessed using PCR and FISH. We confirmed the utility of our panels by establishing the percentage of donor DNA present in the hybrids. Our hybrid resources will allow inexpensive gene mapping and we expect that this technology can be transferred to many other species. Such successes are providing the basis for a new era of mapping tools, in the form of whole genome radiation hybrid panels, and are opening new possibilities for systematic genome analysis in the animal genetics community.

Animals↗
PubMed1998

Cloning, expression and map assignment of chicken prosaposin.

Prosaposin is the precursor of four small glycoproteins, saposins A-D, that activate lysosomal sphingolipid hydrolysis. A full-length cDNA encoding prosaposin from chicken brain was isolated by PCR. The deduced amino acid sequence predicted that, similarly to human and other mammalian species studied, chicken prosaposin contains 518 residues, including four domains that correspond to saposins A-D. There was 59% identity and 76% similarity of human and chicken prosaposin amino acid sequences. The basic three-dimensional structures of these saposins is predicted to be similar on the basis of the conservation of six cysteine residues and an N-glycosylation site. Identity of amino acid sequences was higher among saposins A, B and D than in saposin C. The predicted amino acid sequence of saposin B matched exactly that of purified chicken saposin B protein. The chicken prosaposin gene was mapped to a single locus, PSAP, in chicken linkage group E11C10 and is closely linked to the ACTA2 locus. This confirms the homology between chicken and human prosaposins and defines a new conserved segment with human chromosome 10q21-q24.

Actins↗
PubMed1998

Parameters of the chicken genome (Gallus gallus).

As more information on the chicken genome is gathered, it is becoming increasingly more important to be able to correlate genetic and physical maps. Quantitation of the chicken karyotype is important in establishing parameters which define the genome. Here we report on the physical lengths of the chicken macrochromosomes and establish the DNA content of each, thus identifying implicitly how much of the genome is represented by the microchromosomal component. For the first time, genetic and physical data on the chicken karyotype are presented in relation to one another.

Animals↗
PubMed1997

Improved EBV-based shuttle vector system: dicistronic mRNA couples the synthesis of the Epstein-Barr nuclear antigen-1 protein to neomycin resistance.

Use of EBV-based vector systems has been limited by the requirement to generate EBNA+ cells which are 'permissive' for replication of an oriP-vector. In current constructs, selectable marker and EBNA-1 are not always co-expressed. This is a significant problem since the EBNA-1 gene product can be toxic in some cell types and may be selected against. In this paper, we describe a gene construct that overcomes this limitation. We have exploited the piconaviral internal ribosome entry site to allow the genes for Epstein-Barr nuclear antigen-1 and G-418 resistance to be transcribed as a dicistronic fusion mRNA under the control of the phosphoglucokinase promoter. This construct can be routinely integrated into human cell lines. The presence of EBNA-1 protein was reflected by a large increase in transfection frequencies (1000-fold) using an oriP-based vector which was shown to replicate stably in these cells with no apparent gross rearrangements detected after 8 weeks in culture. Using this system, G-418 resistance should directly reflect integration, as well as expression of the EBNA-1 gene, which, in turn, increases transfection frequencies and stability of EBV-based vector systems and should result in its increased use.

Binding Sites↗
PubMed1997

Gene homologs on human chromosome 15q21-q26 and a chicken microchromosome identify a new conserved segment.

The genes for insulin-like growth factor 1 receptor (IGF1R), aggrecan (AGC1), beta2-microglobulin (B2M), and an H6-related gene have been mapped to a single chicken microchromosome by genetic linkage analysis. In addition, a second H6-related gene was mapped to chicken macrochromosome 3. The Igf1r and Agc1 loci are syntenic on mouse Chr 7, together with Hmx3, an H6-like locus. This suggests that the H6-related locus, which maps to the chicken microchromosome in this study, is the homolog of mouse Hmx3. The IGF1R, AGC1, and B2M loci are located on human Chr 15, probably in the same order as found for this chicken microchromosome. This conserved segment, however, is not entirely conserved in the mouse and is split between Chr 7 (Igf1r-Agc) and 2 (B2m). This comparison also predicts that the HMX3 locus may map to the short arm of human Chr 15. The conserved segment defined by the IGF1R-AGC1-HMX3-B2M loci is approximately 21-35 Mb in length and probably covers the entire chicken microchromosome. These results suggest that a segment of human Chr 15 has been conserved as a chicken microchromosome. The significance of this result is discussed with reference to the evolution of the avian and mammalian genomes.

Aggrecans↗
PubMed1997

Two applications to facilitate the viewing of database search result files on the Macintosh.

MOTIVATION: MacBOB (Macintosh BLAST Output Browser) and MacBOB Filter are two Macintosh-based applications that greatly simplify the viewing of BLAST and FASTA search results files. AVAILABILITY: The programs can be obtained via anonymous ftp from ftp.ri.bbsrc.ac.uk from the directory/pub/software/MacBOB, or via WWW from the Roslin Institute Home Page (http://www.ri.bbsrc.ac.uk/).

Base Sequence↗
PubMed1996

Expression of the gene for transforming growth factor-beta in avian dyschondroplasia.

Previous immunolocalisation studies of dyschondroplasia have indicated that there is a reduction in the number of growth plate chondrocytes containing the protein transforming growth factor beta 3 (TGF-beta 3). The reduction in TGF-beta 3 in dyschondroplasia is likely to be a direct result of a reduction in the expression of the TGF-beta 3 gene. mRNA was extracted from small (0.09 g) samples of growth cartilage from the proximal tibiotarsus of three-week-old broiler chicks. The cartilage samples contained cells from all three zones of the growth plate (proliferative, transitional and upper hypertrophic) and were collected from normal and dyschondroplastic growth plates. The dyschondroplastic growth plates were identified by an accumulation of transitional chondrocytes which were considered to be a result of a failure to differentiate to the hypertrophic phenotype. A semi-quantitative polymerase chain reaction (PCR) was used to estimate the quantity of mRNA specific for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and for each of the three isoforms of TGF beta (TGF-beta 1, TGF-beta 2 and TGF-beta 3) in each of the cartilage samples. The levels of expression of mRNA for GAPDH, TGF-beta 1 and TGF-beta 2 were similar in the two groups, but the expression of TGF-beta 3 mRNA was significantly reduced in the samples from the dyschondroplastic growth plates. The reduction in TGF-beta 3 levels is thought to be associated with the failure of chondrocyte hypertrophy in dyschondroplasia, and provides in vivo evidence that TGF-beta 3 is part of the cascade of events associated with the differentiation of chondrocytes during endochondral ossification in the chick.

Animals↗
PubMed1996

qValue--a program to calculate comparative measures of genomic reorganisation from cytogenetic and/or linkage information.

A program qValue, which calculates a measure of the genomic reorganisation that has occurred between pairs of species since their divergence from a common ancestor, is described. The program takes a tab-delimited text file containing data describing the location of various genetic loci in multiple species and generates an output text file, also in tab-delimited format, that lists various parameters of genomic reorganisation between all possible pairs of species considered. This provides a useful tool for the developing field of comparative genome mapping, particularly in the study of the evolution of the vertebrate genome.

Animals↗
PubMed1996

The expression of transforming growth factor-beta by cultured chick growth plate chondrocytes: differential regulation by 1,25-dihydroxyvitamin D3.

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) and transforming growth factor-beta (TGF-beta) are both important regulators of chondrocyte growth and differentiation. We report here that 1,25(OH)2D3 differentially regulates the expression of the genes for TGF-beta 1 to -beta 3 and the secretion of the corresponding proteins in cultured chick chondrocytes. Confluent growth plate chondrocytes were serum-deprived and cultured in varying concentrations of 1,25(OH)2D3. Cells were assayed for TGF-beta mRNA and conditioned medium was assayed for TGF-beta activity and isoform composition. Active TGF-beta was only detected in 10(-8) M 1,25(OH)2D3-treated cultures (8.37 ng active TGF-beta/mg protein). There was a significant decrease in total (latent-active) TGF-beta activity in conditioned medium of 10(-12) M (23.4%; P < 0.05) and 10(-10) M (20.7%; P < 0.05) 1,25(OH)2D3-treated cultures but 10(-8) M 1,25(OH)2D3 significantly increased (30.9%; P < 0.01) TGF-beta activity. The amounts of TGF-beta 1, -beta 2 and -beta 3 isoforms produced were similar in control, 10(-10) or 10(-12) M 1,25(OH)2D3-treated cultures but the conditioned medium of 10(-8) M 1,25(OH)2D3-treated cultures contained significantly higher amounts of all three isoforms. Quantification of TGF-beta mRNA demonstrated differential control of TGF-beta gene expression with TGF-beta 1 and -beta 3 mRNA levels reduced by all concentrations of 1,25(OH)2D3 examined (10(-8), 10(-10) and 10(-12) M) whilst TGF-beta 2 mRNA concentrations were elevated. Our results indicated that 1,25(OH)2D3 regulates chick growth plate chondrocyte TGF-beta secretion and mRNA expression in a concentration-dependent and isoform-specific manner. This interaction may be important in the regulation of chondrocyte metabolism and endochondral bone growth.

Animals↗
PubMed1995

Expression of genes encoding bone morphogenetic proteins and sonic hedgehog in talpid (ta3) limb buds: their relationships in the signalling cascade involved in limb patterning.

The chicken mutant talpid3 (ta3) has polydactylous limbs with up to 7-8 morphologically similar digits. This lack of antero-posterior polarity in digit pattern is correlated with symmetrical expression of genes of the HoxD complex. We determined the distribution of polarizing activity in limb buds of the chick mutant ta3 by assessing the ability of mesenchyme from various positions along the antero-posterior axis to induce digit duplications when grafted anteriorly into a normal limb. Cells with highest polarizing activity were found at the posterior margin of the wing as in the polarizing region of normal limb buds. However, in contrast to normal limb buds, ta3 anterior mesenchyme also had low polarizing activity. Application of retinoic acid or a polarizing region graft to the anterior of ta3 limb buds changed digit morphology but did not induce digit duplications or digits with any characteristic a-p pattern. To determine which genes are associated with polarizing activity and which are associated with patterning of the digits, we examined expression of the genes Sonic hedgehog (shh), Bmp-2, and Bmp-7, whose expression is normally confined to the posterior margin of the early wing bud and is associated with the polarizing region. In addition, we determined the distribution of Fgf-4 transcripts which in normal limb buds are restricted to the posterior part of the apical ectodermal ridge. In ta3 limb buds, shh expression is restricted to the posterior limb mesenchyme, which has high polarizing activity, but is not expressed in regions which have low polarizing activity. In contrast, Bmp-2 and Bmp-7 are expressed uniformly along the a-p axis. Fgf-4 transcripts are present throughout the apical ectodermal ridge in ta3 limb buds. In the ta3 mutant, there is both an abnormal distribution of signalling activity and response to polarizing signals. In addition, the dissociation between the expression of shh and Bmps suggests distinct roles for the encoded molecules in signalling and response in a-p patterning of limb buds.

Animals↗
PubMed1995

Expression of transforming growth factor-beta mRNA in chicken ovarian follicular tissue.

RNA was isolated from chicken thecal tissue from the largest (F1), third largest (F3), and fifth largest (F5) preovulatory follicles, from small yolky follicles, and from granulosa tissue from F1 follicles. Transforming growth factor-beta (TGF-beta) gene expression was measured using reverse transcription polymerase chain reaction. Thecal cells from all follicle sizes expressed all three isoforms of TGF-beta. TGF-beta 1 mRNA was detected in granulosa cells at levels comparable to those seen in thecal cells. However, TGF-beta 2 and TGF-beta 3 mRNA was expressed at significantly lower levels in granulosa than in thecal tissue. This is the first demonstration of TGF-beta gene expression in the ovary of a nonmammalian species. The similarities between mammalian and avian TGF-beta gene expression are remarkable, especially in light of the distinctive patterns of avian follicular development and the differing steroidogenic capacities of ovarian cell types of the two classes of vertebrates.

Animals↗
PubMed1995

Chicken genome mapping: a new era in avian genetics.

More than 460 loci representing either expressed or anonymous sequences have been mapped on to the first comprehensive molecular genetic linkage map of the chicken genome. Here, we review the current status of poultry genome mapping and discuss some of the new opportunities this provides.

Animals↗
PubMed1995

The chicken transforming growth factor-beta 3 gene: genomic structure, transcriptional analysis, and chromosomal location.

In this paper, we report the isolation, characterization, and mapping of the chicken transforming growth factor-beta 3 (TGF-beta 3) gene. The gene contains seven exons and six introns spanning 16-kb of the chicken genome. A comparison of the 5'-flanking regions of human and chicken TGF-beta 3 genes reveals two regions of sequence conservation. The first contains ATF/CRE and TBP/TATA sequence motifs within an 87-bp region. The second is a 162-bp region with no known sequence motifs. Identification of transcription start sites using chicken RNA isolated from various embryonic and adult tissues reveals two sites of initiation, P1 and P2, which map to these two conserved regions. Comparison of 3'-flanking regions of chicken and mammalian TGF-beta 3 genes also revealed conserved sequences. The most significant homologies were found in the 3'-most end of the transcribed region. DNA sequence analysis of chicken TGF-beta 3 cDNAs isolated by 3'-RACE revealed multiple polyadenylation sites unusually distant from a poly(A) signal motif. A Msc I restriction fragment length polymorphism (RFLP) marker was used to map the TGFB3 locus to linkage group E7 on the East Lansing reference backcross. Linkage to the TH locus showed that the TGFB3 locus was physically located on chicken chromosome 5.

Amino Acid Sequence↗
PubMed1994

Insulin-like growth factor-I in the ovary of the laying hen: gene expression and biological actions on granulosa and thecal cells.

Concentrations of insulin-like growth factor-I (IGF-I) were measured in granulosa and thecal tissue dissected from the three largest follicles in the ovaries of laying hens. The higher concentration was found in extracts of granulosa (0.82 +/- 0.01 pmol/g wet wt) and theca (0.36 +/- 0.02), both of which were greater than that in liver extracts (0.25 +/- 0.01). RNA was extracted from these tissues, and by using reverse transcription and the polymerase chain reaction with primers specific for chicken IGF-I, both granulosa and thecal tissue were shown to express chicken IGF-I mRNA. Granulosa and thecal cell cultures were established and used to measure IGF binding sites and the response to exogenous IGF peptides in terms of DNA synthesis. Both cell types bound [125I]IGF-I, which was displaced by IGF-I, IGF-II, and insulin in descending order of potency, characteristic of a type-I IGF receptor. Treatment of granulosa and thecal cell cultures with IGF-I resulted in a dose-dependent increase in [3H]thymidine incorporation into DNA by both cell types. LH, but not FSH, stimulated DNA synthesis in cultured granulosa cells but not in cultured thecal cells. This effect was enhanced in granulosa cells by the addition of IGF-I to the culture medium. These data are consistent with an autocrine or paracrine role for IGF-I within the developing ovarian follicle of the domestic hen.

Animals↗
PubMed1994

Tissue specific expression of an alpha-skeletal actin-lacZ fusion gene during development in transgenic mice.

Transgenic mice carrying a chimaeric transgene containing 730 bp of the 5'-flanking sequences and the entire first intron of the rat alpha-skeletal actin gene fused to the lacZ reporter gene have been produced by microinjection. The lacZ reporter gene was used to verify the suitability of using the rat alpha-actin promoter elements to target expression of genes of agricultural and therapeutic value exclusively to skeletal and heart muscle cells and fibres of transgenic mice. Expression of the transgene indicates a tightly regulated developmental and muscle specific control of the rat alpha-skeletal actin gene, making it a useful promoter for gene targeting to muscle tissues. The cells destined to form muscle tissues in these transgenic mice are readily visualized in intact embryos by staining for beta-galactosidase activity, making them a suitable animal model for studying the origin and development of skeletal and cardiac muscle tissues.

Actins↗
PubMed1994

Evolution of the transforming growth factor-beta superfamily.

Transforming growth factor beta 1 (TGF-beta 1) is the prototype of an increasingly complex superfamily of growth and differentiation factors. To date, a total of 74 TGF-beta-like sequences have been published, probably representing 23 distinct genes. These sequences were obtained from mammalian, avian, amphibian and insect species, thus emphasising the ancient nature of the TGF-beta superfamily peptides. This article summarises current hypotheses concerning the evolutionary history of this protein superfamily, based on the molecular phylogeny of the published sequences. Comparison of the deduced amino acid sequences leads to the definition of five main groups within the superfamily (TGF-beta, Bone Morphogenetic Proteins [BMP], Anti-Müllerian Hormone [AMH], Inhibin alpha [INH alpha] and GDF-9) and six subgroups within the BMPs (60A, Decapentaplegic [dpp], Vg1, BMP-3, Inhibin beta [INH beta A/B] and nodal). This classification predicts possible phylogenetic and functional relationships among these proteins.

Amino Acid Sequence↗