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C K Tuggle

Publications and source records attributed to C K Tuggle.

At least 37 records · Page 2Linked to original sources
PubMed2001

Cloning of the full length pig PIT1 (POU1F1) CDNA and a novel alternative PIT1 transcript, and functional studies of their encoded proteins.

PIT1 is an essential regulatory gene of growth hormone (GH), prolactin (PRL) and thyrotropin beta subunit (TSHbeta). Previously, a partial pig PIT1 cDNA and a genomic clone of the entire 3' end of the PIT1 gene was isolated, and polymorphisms at PIT1 were associated with several performance traits in the pig. In order to understand the biological function of the pig PIT1 gene and its possible application in swine genetics, reverse transcriptase-polymerase chain reaction (RT-PCR) was used to complete the cloning of the full length cDNA for pig PIT1. The pig PIT1 cDNA and its deduced protein sequence have approximately 90% and 95% identity, respectively, with the PIT1 cDNA and protein of other mammals (human, bovine, sheep and rodents). Surprisingly, sequence comparison to other pig PIT1 sequences indicated only approximately 93% identity. Additional sequencing confirmed our sequence, and identified a new polymorphism in exon 4. Phylogenetic analysis of several mammalian PIT1 sequences indicates sequencing errors may account for the discrepancies observed in the other pig sequences reported. Several PIT1 alternative spliced forms were also identified by RT-PCR. They were the delta3PIT1 (missing entire exon 3), delta4PIT1 (missing entire exon 4) and PIT1beta (additional 26 amino acids inserted in front of exon 2) transcripts. The delta4PIT1 and PIT1beta transcripts have been found to encode functionally different proteins in rodents. The delta3PIT1 transcript is a novel isoform of PIT1. Potentially different functions between pig delta3PIT1 and PIT1 were analyzed by expressing these proteins in bacteria. The E. coli-expressed PIT1 and delta3PIT1 proteins were used with rat growth hormone (rGH) and rat prolactin (rPRL) promoter DNA in DNA mobility shift assays. The results showed that pig PIT1 can specifically bind rGH and rPRL promoter regions, but that the pig delta3PIT1 cannot, even at very high protein concentrations. Possible protein-protein interactions between delta3PIT1 and PIT1 were tested by mixing protein extracts before the gel shift assay, and the results showed that delta3PIT1 protein did not affect PIT1 binding to its target DNA. These data demonstrate the functionality of the PIT1 cDNA cloned in this study, and identify a novel delta3PIT1 transcript which encodes a protein that cannot bind rGH/rPRL target sequences.

Amino Acid Sequence↗
PubMed2000

Mapping of the CYP1A1, SSTR1 and TTF1 genes to pig chromosome 7q refines the porcine-human comparative map.

The cytochrome P-450, subfamily I, polypeptide 1 (CYP1A1), somatostatin receptor 1 (SSTR1) and transcription termination factor 1 (TTF1) genes, predicted to reside on SSC7q based on chromosome painting studies, were selected for genetic mapping in the pig. Porcine sequence tagged sites (STSs) spanning 0.9-1.5 kb genomic DNA sequences were generated and all genes were physically assigned to SSC7q12-q23, q26 using a pig-rodent somatic cell hybrid panel. Direct sequencing of PCR products from four different breeds revealed single nucleotide polymorphisms (SNPs) in all genes. SNPs in the SSTR1 and CYP1A1 genes allowed simple genotyping using PCR-restriction fragment length polymorphism (RFLP) analysis and linkage mapping with the PiGMaP reference families. SSTR1 was found to be closely linked to CYP1A1 (0.0 cM; LOD = 11.1) and ANPEP (0.0 cM; LOD = 9.0). This refined the localization of a previously identified breakpoint in the linkage map between regions with orthology to human chromosome 15q (represented by CYP1A1 and ANPEP) and 14q (represented by SSTR1). Moreover, multipoint linkage data suggest inverted segments of orthology between HSA14q11-q13 and SSC7q. The TTF1 SNP was confirmed by allele-specific PCR but was not informative in any of the families used for linkage analysis.

Animals↗
PubMed1999

Hoxa5 gene regulation: A gradient of binding activity to a brachial spinal cord element.

The Hox genes cooperate in providing positional information needed for spatial and temporal patterning of the vertebrate body axis. However, the biological mechanisms behind spatial Hox expression are largely unknown. In transgenic mice, gene fusions between Hoxa5 (previously called Hox-1.3) 5' flanking regions and the lacZ reporter gene show tissue- and time-specific expression in the brachial spinal cord in day 11-13 embryos. A 604-bp regulatory region with enhancer properties directs this spatially specific expression. Fine-detail mapping of the enhancer has identified several elements involved in region-specific expression, including an element required for expression in the brachial spinal cord. Factors in embryonic day 12.5 nuclear extracts bind this element in electrophoretic mobility shift assays (EMSA) and protect three regions from DNase digestion. All three sites contain an AAATAA sequence and mutations at these sites reduce or abolish binding. Furthermore, this element binds specific individual embryonic proteins on a protein blot. The binding activity appears as a gradient along the anterior-posterior axis with two- to threefold higher levels observed in extracts from anterior regions than from posterior regions. In parallel with the EMSA, the proteins on the protein blot also show reduced binding to probes with mutations at the AAATAA sites. Most importantly, transgenic mice carrying Hoxa5/lacZ fusions with the three AAATAA sites mutated either do not express the transgene or have altered transgene expression. The brachial spinal cord element and its binding proteins are likely to be involved in spatial expression of Hoxa5 during development.

Animals↗
PubMed1999

New insights into porcine-human synteny conservation.

Eleven genes were mapped to the porcine genome with the aim of improving the human-porcine comparative gene map. Five of these genes were from regions of the human genome painted by porcine chromosomal probes; of these, two mapped to chromosomes not expected from the painting results. Among the six genes from human regions not painted by porcine chromosomal probes, three genes did not map where expected by the principle of parsimony. Several of the gene assignments indicate the existence of small regions of conserved synteny not detected by heterologous chromosome painting, especially in telomeric regions. We have also detected new rearrangements in gene order within the regions of correspondence between human Chromosome (HSA) 15 and porcine Chromosome (SSC) 1 as well as between HSA4 and SSC8.

Animals↗
PubMed1999

Physical assignments of human chromosome 13 genes on pig chromosome 11 demonstrate extensive synteny and gene order conservation between pig and human.

Previous mapping between the human and pig genomes suggested extensive conservation of human chromosome 13 (HSA13) to pig chromosome 11 (SSC11). The objectives of this study were comparative gene mapping of pig homologs of HSA13 genes and examining gene order within this conserved synteny group by physical assignment of each locus. A detailed HSA13 to SSC11 comparison was chosen since the comparative gene map is not well developed for these chromosomes and a rearranged gene order within conserved synteny groups was observed from the comparison between HSA13 and bovine chromosome 12 (BTA12). Heterologous primers for PCR were designed and used to amplify pig homologous fragments. The pig fragments were sequenced to confirm the homology. Six pig STSs (FLT1, ESD, RB1, HTR2A, EDNRB, and F10) were physically mapped using a somatic cell hybrid panel to SSC11, and fluorescent in situ hybridization (FISH) mapping was also applied to improve map resolution and determine gene order. Results from this study increase the comparative information available on SSC11 and suggest a conserved gene order on SSC11 and HSA13, in contrast to human:bovine comparisons of this syntenic group.

Animals↗
PubMed1999

Tissue expression patterns of chicken octamer-binding proteins.

The octamer motif is important in transcriptional regulation of genes of the immune system in many species, including the chicken. Little is known, however, regarding octamer-binding protein expression in chicken tissues. We examined octamer-binding protein expression patterns in multiple chicken tissues (ovary, cerebrum, liver, lung, kidney, spleen, thymus, and bursa of Fabricius) plus two lymphocyte cell lines. Every tissue and cell line had multiple octamer-binding proteins. Seven distinct protein-DNA complexes were identified. Our results demonstrate that multiple octamer-binding proteins, exhibiting differential tissue expression, exist in a wide variety of tissues of the chicken.

Animals↗
PubMed1999

Human chromosome 3 and pig chromosome 13 show complete synteny conservation but extensive gene-order differences.

A comparative map of human chromosome 3 (HSA 3) and pig chromosome 13 (SSC 13) was constructed using physically assigned pig sequence-tagged sites (STSs). Pig STSs representing 11 HSA 3 genes, including v-Raf-1 murine leukemia viral oncogene homolog 1 (RAF1), retinoic acid beta receptor (RARB), cholecystokinin (CCK), pituitary transcription factor 1 (POU1F1), ceruloplasmin (CP), guanine nucleotide binding protein, alpha-inhibiting polypeptide 2 (GNAI2), sucrase-isomaltase (SI), rhodopsin (RHO), dopamine receptor D3 (DRD3), growth-associated protein 43 (GAP43), and somatostatin (SST), were developed. Ten pig STSs were regionally mapped using a somatic cell hybrid panel (SCHP) to SSC 13 with 80-100% concordance. Large-insert probes were obtained by screening a pig yeast artificial chromosome (YAC) library with primers for each STS. Several YACs were identified for DRD3, GAP43, POU1F1, RHO, SI, and SST for fluorescence in situ hybridization (FISH) mapping. Single gene and bi-color FISH with each pairwise combination were used to further define the gene order on SSC 13. While these data confirm chromosome painting results showing that HSA 3 probes hybridize to a major portion of SSC 13, they also demonstrate extensive gene-order differences between man and pig within this large conserved synteny group. Interestingly, several conserved chromosomal regions have been detected between pig and mouse that are not conserved between man and mouse, suggesting that the SSC 13 gene arrangement may be the closest to that of the ancestral eutherian chromosome.

Animals↗
PubMed1998

Linkage and physical mapping of prolactin to porcine chromosome 7.

Comparative mapping studies between human and pig have shown that there is conserved synteny between human chromosome 6 and pig chromosomes 1 and 7, but some gene locations are not well established. Prolactin (PRL), an anterior pituitary hormone, has been mapped to human chromosome 6, and has tentatively mapped to pig chromosome 7 using Southern-RFLP analysis with a limited number of meioses. To confirm the assignment of prolactin to porcine chromosome 7 by physical and linkage analysis, pig cDNA and human genomic DNA sequences were used to design pig-specific PCR primers. The primers amplified a fragment of approximately 2.8 kb. Two polymorphic restriction sites were identified within this fragment with the restriction endonuclease BstUI. Prolactin was significantly linked to six markers on the published PiGMaP map of pig chromosome 7. Prolactin was physically mapped using a pig x rodent somatic cell hybrid panel. An analysis of these data placed PRL on pig 7p1.1-p1.2 with 100% concordance and was in complete agreement with the linkage data. Both mapping techniques placed PRL in a conserved order with the loci in the syntenic region of human chromosome 6.

Animals↗
PubMed1998

Mapping of the natural resistance-associated macrophage protein 1 (NRAMP1) gene to pig chromosome 15.

The natural resistance-associated macrophage protein 1 (NRAMP1) was mapped in the pig for study as a potential candidate gene in controlling pig resistance to Salmonella infection. Primers were designed from the pig cDNA to amplify a 1.6 kb fragment between exons 1 and 3. By using a pig-rodent somatic cell hybrid panel, NRAMP1 was mapped to pig chromosome 15 (SSC15) with 100% probability, and the regional assignment was SSC15q23-26 with 87% concordance. A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) marker was developed by using the HinfI enzyme and three alleles were identified from a population including 11 breeds. Linkage analysis confirmed the physical assignment by using the PiGMaP reference families. Pig NRAMP1 was linked to SSC15 markers S0088, S0149 and S0284 (LOD > 3). A small population study revealed large allele frequency differences among tested breeds. An A allele is only observed in dam (white) lines whereas a similar exclusivity of the C allele was seen in sire (colored) breeds.

Alleles↗