Avian keratin genes. I. A molecular analysis of the structure and expression of a group of feather keratin genes.

The nucleotide sequence of the four complete chicken feather keratin genes A to D contained in the previously isolated recombinant lambda CFK1 has been determined. All four genes have a very similar structure; each gene encodes a polypeptide of 97 amino acid residues and contains an intron in the 5' non-coding region, 37 base-pairs from the cap site. Comparison of the previously determined feather keratin gene C sequence to genes A, B and D indicates that a high level of gene correction has occurred in the protein coding and 5' non-coding regions, which show more than 90% homology, whereas the intron and 3' non-coding regions are by contrast poorly conserved with one or two exceptions. The dramatic conservation of the 5' non-coding region between the feather keratin sequences and an unrelated but co-expressed gene encoding a histidine-rich protein suggests that this segment may play an important role in transcriptional regulation. In addition, both gene types contain an identically positioned intron in the 5' non-coding region. Northern blots performed using gene-specific probes show that the four characterized genes A to D plus gene E, which is partially contained in the recombinant lambda CFK1, are all expressed in feather tissue from 14-day old chick embryos. In addition, we report that a scale keratin gene (originally isolated from a scale complementary DNA library) is expressed at a low level in the embryonic feather.

Avian keratin genes. II. Chromosomal arrangement and close linkage of three gene families.

We describe the isolation and characterization of a set of overlapping cosmid clones that contain chicken keratin genes. The 100 kb (1 kb = 10(3) base-pairs) of DNA represented in these clones contains a cluster of 18 feather keratin genes spanning 53 kb of DNA. The feather keratin genes are spaced about 3 kb apart and at least 11 of them have the same transcriptional orientation. Southern analysis using oligonucleotide probes made from highly conserved portions of the 5' non-coding, intron and 3' non-coding regions, respectively, indicate that these sequences have been highly conserved among the gene family as a whole, with only one or two exceptions in each case. The presence of some regularly repeated restriction enzyme sites are indicative of tandem duplication events in the recent history of the feather keratin gene family. The feather keratin gene locus is flanked on both sides by related types of keratin genes. On the 5' side of the feather gene cluster are three keratin genes (designated feather-like) that are located 5 kb from the last feather keratin gene and are spaced about 4 kb apart. On the 3' side of the feather gene cluster, 21 kb from the last feather keratin gene, lies a cluster of four genes that encode claw keratins. These genes are spaced about 1 kb apart and appear to be divergently transcribed. Partial DNA sequence analysis of the feather-like gene lying proximal to the feather keratin gene cluster demonstrated that it encodes a protein of 115 amino acid residues that is 80% homologous to the feather keratins at both the DNA and amino acid sequence levels. The feather-like gene(s) are expressed in both embryonic and adult (post-hatch) chick feathers and at a very low level in embryonic scale tissue. These genes therefore form a new family of feather proteins that is distinct from the previously characterized feather keratins.

Investigation of the significance of heavy metal contents of blackbird feathers.

The lead, cadmium and zinc in untreated blackbird (Turdus merula L.) feathers is predominantly of exogenous origin. The endogenous concentration is of minor importance. The degree of surface metal pollution depends on exposure time. The exogenous fraction of heavy metals cannot be completely removed by washing procedures. The difference between washed and unwashed feathers is demonstrated by SEM micrographs.

A survey of polar and nonpolar lipids from epidermis and epidermal appendages of the chicken (Gallus domesticus).

Total lipid was extracted from chicken (Gallus domesticus) epidermis, leg scale, claws, feathers and preen glands and analyzed by quantitative thin-layer chromatography. All of the tissue lipids contained large proportions of wax diesters, triglycerides, and free sterols and variable proportions of phospholipids, steryl esters and free fatty acids. All of the keratinized tissues, but not the preen gland, contained ceramides, acylceramides and cholesteryl sulfate. Acylglucosylceramides were found only in full thickness epidermis. Glucosylsterols and acylglucosylsterols were found in the keratinized tissues, and may be of significance in the evolutionary history of the epidermal water barrier.

Genes for hair and avian keratins.

This paper briefly reviews the present level of understanding of the genes that code for hair keratins and the keratins of avian feather and scale. The emphasis to date has been on the structure of the genes, the derivation of amino acid sequences for several of the proteins from the coding sequences, and the organization of the different gene families within genomic DNA. Genomic sequences for the proteins of the three main gene families for wool keratin have been isolated from sheep genomic libraries and their detailed analysis by DNA sequencing is proceeding. Already, representative sequences for the genes of the microfibril (alpha-filament or IF) proteins and for the cysteine-rich and HGT matrix proteins are available. With these sequences as sources of probes, it is now possible to move from organizational and structural studies and undertake the study of the control of the expression of these genes either in wool (hair) follicles in vivo or after transfection with specific keratin genes of suitable epidermal cells in culture. Parallel with these studies is the investigation of the fate of trichohyalin droplets in the inner root sheath during its maturation, which is coordinated with keratinization in the hair itself. These unusual proteins and the changes they undergo suggest a highly specific function. The discovery of that function might come from the use of molecular cloning techniques especially for the synthesis of quantities of protein adequate for study. The avian keratins of feather and scale are coded for by a large family of evolutionarily related genes as shown in recent investigations of the DNA sequences. Again, derived protein sequences have provided a view of the feather and scale structures previously unavailable. Information on the fine structure of the genes is enabling the examination of their expression in vivo during embryonic development and the possible significance in this development of the histidine-rich ("fast") protein. The lack of any detectable transcripts from characterized feather keratin genes injected into Xenopus oocytes is presenting the possibility of testing for tissue-specific protein factors that might be responsible for the specific activation of keratin genes.

Furazolidone residues in chicken and swine tissues after feeding trials.

Broiler chickens and swine fed furazolidone in their diet were sacrificed, and samples of liver, kidney, skin/fat and muscle were harvested and analyzed for furazolidone residue. Chickens fed 200 g of furazolidone/ton of feed were withdrawn from treatment 21, 14, 7, 5, 3, or 0 days before slaughter. Birds withdrawn from medication more than 5 days prior to slaughter had no residues in any of the tissues sampled. One of the 12 birds in each of the 5 day and 3 day withdrawal groups had detectable residues in the skin/fat. Seven of the 12 birds in the 0 day withdrawal group had residues of less than 2 ppb in skin/fat samples. Chickens fed 400 g furazolidone/ton of feed were withdrawn from treatment 0 days before slaughter. Residues of 0.7 to 3.5 ppb were found in the skin of these birds; residues were not found in other tissues. Swine were fed 300 g furazolidone/ton of feed for 2 weeks or 150 g/ton for 5 weeks. They were withdrawn from treatment 10, 7, 5, 3, or 0 days before slaughter. Tissue samples taken from these swine did not contain detectable furazolidone residues.

Maintenance nitrogen requirement of the turkey breeder hen with an estimate of associated essential amino acid needs.

Nonproducing, small-type breeder hens in excess of 65 weeks of age were used to represent the maintenance state. All birds had been in laying cages since 30 weeks and accustomed to 16 hr of 70 lx lighting at 16 C. Nitrogen (N) balance was performed in metabolism cages under the same conditions. Ad libitum intake of a common breeder ration led to an intake of ca. 47 kcal metabolizable energy (ME)/kg body weight (BW)/day, which was considered to represent the maintenace energy requirement. Nitrogen retained while consuming this feed averaged 172 mg/kg BW/day. Force-feeding a N-free diet to satisfy the maintenance energy requirement resulted in an 85 mg N/kg BW/day endogenous loss. Total maintenance nitrogen requirement was considered to approximate 257 mg/kg BW/day. Nitrogen retention after force-feeding corn-soybean meal rations having a progressive protein content indicated that the associated amino acids were more efficient in satisfying the endogenous than the total N requirement. A model that estimated maintenance amino acid requirements was assembled by combining the relative concentrations found in muscle and feathers to represent endogenous and retained N, respectively. For the most part, model values agreed with published results for the rooster; however, verification in balance studies was less than successful and believed to be attributable to hen variation in feather cover and protein reserves.

Investigations of trace metals in long-tailed duck (Clangula hyemalis L.) from the Gdansk Bay.

The determination of iron, zinc, manganese, copper, lead, cadmium, cobalt and nickel was carried out on liver, breast muscle, heart, stomach and feathers of 50 male and 40 female long-tailed ducks (Clangula hyemalis L.) staying in their winter quarters in Gdansk Bay during 1980-81. There were no significant differences in the concentration of metals between male and female long-tailed ducks. The correlation coefficients between the concentration of metals in the liver and breast muscle were determined.

Amino-acid sequence of feather keratin from fowl.

The amino acid sequence of a single polypeptide chain, B-4, from fowl feather barbs has been determined. The B-4 chain was found to consist of 96 amino acid residues and to have a molecular weight of 10206 in the S-carboxymethylated form. The N terminus of this protein was an N-acetylserine residue. The B-4 protein contained seven S-carboxymethylcysteine residues, six of which are located in the N-terminal region (residues 1-26), and other one in C terminus. The central region of the peptide chain was rich in hydrophobic residues. There were homologous amino acids at 66 positions in the sequences of the feather keratins of fowl, emu and silver gull. The variation (substitution, deletion and insertion) in sequence was found to be localized in both terminal sections of the polypeptide chain. The B-4 protein structure was predicted to contain beta-sheet (about 30%), turn and random-coil-like structure, and no alpha-helix. beta-Sheet structure is mostly located in the central region (residues 22-70). On the other hand, both terminal regions are almost devoid of secondary structure.

[Methodological studies of protein metabolism and the bioenergetics of protein retention in growing animals. 2. The results of amino acid analysis after the long-term labelling of chicks with 15NH4-acetate]. / Methodische Untersuchungen zum Eiweissumsatz und zur Bioenergetik des Eiweissansatzes bei wachsenden Tieren. 2. Mitteilung. Ergebnisse der Aminosäurenanalytik nach Langzeitmarkierung von Küken mit 15NH4-Azetat.

The amino acid composition of the proteins in selected body fractions of chickens and the 15N-excess of amino acids isolated from them resulting from a feeding experiment with long-term labelling with 15NH4-acetate supplied orally are given. The amino acid spectra of feathers, breast and leg muscles are characterised by differences in the content of individual amino acids specific for the organs, the composition of the proteins, however, is independent of the protein content of the ration and the age of the animals. The sarcoplasmatic and myofibrillar proteins also have typical amino acid patterns, which--with the exception of the histidine content--are neither influenced by the extraction of the proteins from the breast or leg muscles nor by the energy level of the feeding or the age of the animals. There are no significant differences in the metabolisation of the main protein fraction of the breast and leg muscles. The oral supply of 15N ammonium acetate to broilers predominantly labels the non-essential amino acids so that the derived kinetic data chiefly represent the metabolism of the non-essential amino acids.