Identifying genes involved in the variability of genetic fatness in the growing chicken.

A precise knowledge of the genome involved in the expression of a quantitative trait could provide a useful tool in breeding programs; molecular genetic methods are capable of yielding this kind of information. An experimental procedure is presented here for identifying genes whose expression is related to weight variability of abdominal adipose tissue in the growing chicken. Quantitative traits are the result of metabolic pathways exhibiting some major regulation stages that are controlled genetically. These steps involve genes that may act as "major genes". With regard to chicken fat metabolism, most fatty acids are synthesized in the liver and incorporated into very low density lipoprotein (VLDL) particles before their secretion into the plasma. Accordingly, the present study focused on the expression of liver genes. The mRNA of lipogenic enzymes (acetyl-coenzyme-A carboxylase, fatty acid synthase, malic enzyme, and delta 9-desaturase) were analyzed. Also studied were apoprotein (apo)A1, apoVLDL-II, and apoB mRNA from 9-wk-old male chickens from two lines selected for high and low abdominal fat pads. Significant differences for apoA1 mRNA levels occurred between fat and lean birds. Moreover, the total quantity of mRNA provided an accurate estimation of the abdominal fat pad (r = .74 with P < .05).

Cloning and characterization of the 5' end and promoter region of the chicken acetyl-CoA carboxylase gene.

Acetyl-CoA carboxylase is a rate-limiting enzyme in the biogenesis of long-chain fatty acids. In the present study, the 5' end and flanking region of the acetyl-CoA carboxylase (ACC) gene was analysed in the chicken. A genomic clone was isolated containing the first three exons, the third one containing the ATG codon. Using nuclease-mapping experiments and primer-extension analyses, the transcription-initiation site was located 153 nucleotides upstream of the ATG codon. In contrast with rat ACC gene expression, reverse transcriptase PCR analysis performed on chicken liver mRNA did not reveal alternative splicing in the 5'-untranslated region of these messengers. The promoter region is very G+C rich, and contains no TATA or CAAT boxes. Analysis by transient transfection in a human hepatoma cell line (HepG2) demonstrates that the promoter activity requires the presence of symmetrical sequences located upstream of the GC boxes. Transcription of this gene is found to be controlled by tri-iodothyronine in HepG2 cells, but the sequence responsible for the tri-iodothyronine response is not the consensus tri-iodothyronine-responsive element localized in the promoter. These results bring new insights to the regulation of the chicken ACC gene which differs from that of the rat.

Characterization of the chicken fatty acid synthase gene 5' part and promoter region.

Fatty acid synthase activity has been shown to be regulated mainly at the transcriptional level under both dietary and hormonal influences. As a first step towards elucidating the factors involved, we isolated and characterized chicken genomic clones encompassing the 5' part of the chicken fatty acid synthase gene and its flanking region. The entire region of the cloned DNA spans 30 kb, and the first three exons of the gene were mapped to a 6.3-kb genomic fragment. The transcription initiation site was determined after subcloning the cDNA which encodes the 5' end of the mRNA. The first exon, which was 129 bp long, was located approximately 5.3 kb upstream of the second exon, which contained the start codon. In the 5' flanking region, putative TATA and CAAT boxes were located 30 and 92 bp, respectively, upstream of the transcription initiation site. The 5' flanking region contained numerous sequences corresponding to consensus binding sites for transcription factors. Various lengths of flanking sequences extending up to 1028 bp upstream of the transcription initiation site and containing 100 bp of the first exon were linked to the bacterial chloramphenicol acetyltransferase gene; in this study, these constructs were analyzed in transient transfection assays in human hepatoma cells. The proximal 125-bp sequence upstream of the transcription start site was shown to be a basal promoter. The cloning and characterization of the chicken fatty-acid synthase gene provides some further insight into the regulation of fatty acid synthesis in birds as compared to mammals.

Malic enzyme gene in chick embryo hepatocytes in culture: clofibrate regulates responsiveness to triiodothyronine.

In chick embryo hepatocytes, triiodothyronine (T3) causes a 30- to 40-fold increase in malic enzyme activity when added between 1 and 3 days, but has no effect when added between 5 and 7 days in culture. This transcription-mediated decline in T3 responsiveness is partially reversed by corticosterone (Roncero, C. and A. G. Goodridge, 1992. Arch. Biochem. Biophys. 295: 258-267). Clofibrate also reversed the decline in responsiveness to T3, and did so in the absence of an increase in binding of T3 to nuclear receptors. The effects of clofibrate and corticosterone were additive, suggesting different mechanisms. The responsiveness of a gene to a specific agent depends on specific regulatory sequences of DNA in that gene. When 5.8 kb of the 5'-flanking DNA of the malic enzyme gene was linked to the chloramphenicol acetyltransferase (CAT) gene and transfected into hepatocytes, T3 stimulated CAT activity. Responsiveness of CAT activity to T3 decreased with time, and this decrease was partially reversed by clofibrate. The T3 responses of cells transfected with various chimeric DNAs that contained T3 response elements (T3REs) of the malic enzyme gene or synthetic consensus T3REs also were increased by clofibrate. The results suggest that clofibrate regulates expression of a metabolite or a protein factor which, in turn, influences function of the T3 receptor.

Mapping of FASN and ACACA on two chicken microchromosomes disrupts the human 17q syntenic group well conserved in mammals.

Fatty acid synthase and Acetyl-CoA carboxylase are both key enzymes of lipogenesis and may play a crucial role in the weight variability of abdominal adipose tissue in the growing chicken. They are encoded by the FASN and ACACA genes, located on human Chromosome (Chr) 17q25 and on Chr 17q12 or 17q21 respectively, a large region of conserved synteny among mammals. We have localized the homologous chicken genes FASN and ACACA coding for these enzymes, by single-strand conformation polymorphism analysis on different linkage groups of the Compton and East Lansing consensus genetic maps and by FISH on two different chicken microchromosomes. Although synteny is not conserved between these two genes, our results revealed linkage in chicken between FASN and NDPK (nucleoside diphosphate kinase), a homolog to the human NME1 and NME2 genes (non-metastatic cell proteins 1 and 2), both located on human Chr 17q21.3, and also between FASN and H3F3B (H3 histone family 3B), located on human Chr 17q25. The analysis of mapping data from the literature for other chicken and mammalian genes indicates rearrangements have occurred in this region in the mammalian lineage since the mammalian and avian radiation.