Expression of a wool intermediate filament keratin transgene in sheep fibre alters structure.

Alteration of the protein composition of the wool fibre via transgenesis with sheep wool keratin and keratin associated protein (KAP) genes may lead to production of fibre types with improved processing and wearing qualities. Using this approach, we have demonstrated that high level cortical-specific expression of a wool type II intermediate filament (IF) keratin gene, K2.10, leads to marked alterations in both the microstructure and macrostructure of the wool fibres, which have higher lustre and reduced crimp. Analysis of mRNA found reduced levels of transcripts from endogenous cortical type I (p < 0.05) and type II (p < 0.01) keratin IF genes and from the KAP8 (p < 0.001) and KAP2 (p < 0.01) gene families. Examination of protein composition revealed an altered ratio in the keratin type II protein family of the wool fibre cortex. Whilst the over-expressed K2.10 transgene product constituted the majority of keratin type II IF protein, it appeared unable to form heterodimers with much of the expressed endogenous keratin type I IF. In comparison with non-transgenic sheep, fewer IF microfibrils were visible in the cortical cells of fibres from transgenics. The combined effect on fibre structure was disruption of the formation of orthocortical and paracortical cells in the fibre cortex, a factor which could account for the reduction in fibre crimp. No effects upon transcript or protein levels, or fibre microstructure or macrostructure were observed in transgenic sheep expressing the transgene at lower levels, indicating that subtle changes to the gene expression profile in sheep wool follicles can be tolerated. The data here also illustrate that control over endogenous transcript levels in the cortex results when factors acting on the endogenous keratin type I, keratin type II and KAP gene sequences are sequestered by the active K2.10 transgene locus. Moreover, interference to a transcriptional hierarchy shared by keratin and KAP genes may occur prior to establishment of the orthocortical and paracortical compartments of the follicle cortex, at the level of the chromatin.

Regulation of a hair follicle keratin intermediate filament gene promoter.

During hair growth, cortical cells emerging from the proliferative follicle bulb rapidly undergo a differentiation program and synthesise large amounts of hair keratin proteins. To identify some of the controls that specify expression of hair genes we have defined the minimal promoter of the wool keratin intermediate filament gene K2.10. The region of this gene spanning nucleotides -350 to +53 was sufficient to direct expression of the lacZ gene to the follicle cortex of transgenic mice but deletion of nucleotides -350 to -150 led to a complete loss of promoter activity. When a four base substitution mutation was introduced into the minimal functional promoter at the binding site for lymphoid enhancer factor 1 (LEF-1), promoter activity in transgenic mice was decreased but specificity was not affected. To investigate the interaction of trans-acting factors within the minimal K2.10 promoter we performed DNase I footprinting analyses and electrophoretic mobility shift assays. In addition to LEF-1, Sp1, AP2-like and NF1-like proteins bound to the promoter. The Sp1 and AP2-like proteins bound sequences flanking the LEF-1 binding site whereas the NF1-like proteins bound closer to the transcription start site. We conclude that the LEF-1 binding site is an enhancer element of the K2.10 promoter in the hair follicle cortex and that factors other than LEF-1 regulate promoter tissue- and differentiation-specificity.

The role of keratin proteins and their genes in the growth, structure and properties of hair.

The importance of wool in the textile industry has inspired extensive research into its structure since the 1960s. Over the past several years, however, the hair follicle has increased in significance as a system for studying developmental events and the process of terminal differentiation. The present chapter seeks to integrate the expanding literature and present a broad picture of what we know of the structure and formation of hair at the cellular and molecular level. We describe in detail the hair keratin proteins and their genes, their structure, function and regulation in the hair follicle, and also the major proteins and genes of the inner and outer root sheaths. We discuss hair follicle development with an emphasis on the factors involved and describe some hair genetic diseases and transgenic and gene knockout models because, in some cases, they stimulate natural mutations that are advancing our understanding of cellular interactions in the formation of hair.

Expression of bacterial cysteine biosynthesis genes in transgenic mice and sheep: toward a new in vivo amino acid biosynthesis pathway and improved wool growth.

It is possible to improve wool growth through increasing the supply of cysteine available for protein synthesis and cell division in the wool follicle. As mammals can only synthesise cysteine indirectly from methionine via trans-sulphuration, expression of transgenes encoding microbial cysteine biosynthesis enzymes could provide a more efficient pathway to cysteine synthesis in the sheep. If expressed in the rumen epithelium, the abundant sulphide, produced by ruminal microorganisms and normally excreted, could be captured for conversion to cysteine. This paper describes the characterisation of expression of the cysteine biosynthesis genes of Salmonella typhimurium, cysE, cysM and cysK, and linked cysEM, cysME and cysKE genes as transgenes in mice and sheep. The linked transgenes were constructed with each gene driven by a separate promoter, either with the Rous sarcoma virus long terminal repeat (RSVLTR) promoter or the mouse phosphoglycerate kinase-1 (mPgk-1) promoter, and with human growth hormone (hGH) polyadenylation sequences. Transgenesis of mice with the RSVLTR-cysE gene afforded tissue-specific, heritable expression of the gene. Despite high levels of expression in a number of tissues, extremely low levels of expression occurred in the stomach and small intestine. Results of a concurrent sheep transgenesis experiment using the RSVLTR-cysEM and -cysME linked transgenes revealed that the RSVLTR promoter was inadequate for expression in the rumen. Moreover, instability of transgenes containing the RSVLTR sequence was observed. Expression of mPgk-cysME and -cysKE linked transgenes in most tissues of the mice examined, including the stomach and small intestine, suggested this promoter to be a better candidate for expression of these transgenes in the analogous tissues of sheep. However, a subsequent sheep transgenesis experiment indicated that use of the mPgk-1 promoter, active ubiquitously and early in development, may be inappropriate for expression of the cysteine biosynthesis transgenes. In summary, these results indicate that enzymically active bacterial cysteine biosynthesis gene products can be coexpressed in mammalian cells in vivo but that expression of the genes should be spatio-temporally restricted to the adult sheep rumen epithelium.

Dietary cysteine regulates the levels of mRNAs encoding a family of cysteine-rich proteins of wool.

The abomasal or intravenous infusion of sulphur-containing amino acids such as cysteine or methionine into sheep on low-quality diets increases the sulphur content of the wool by increasing the synthesis of proteins containing a cysteine content of approximately 30 mol %. To investigate the molecular and cellular basis of this nutritional effect, quantitative analyses of wool keratin mRNA and protein levels, and follicle cortical cell type, were undertaken in sheep intravenously infused with cysteine. Northern blot analyses revealed that the mRNA levels of one gene family encoding cysteine-rich keratin-associated proteins (KAP4 family) expressed in the wool follicle cortex, increased approximately 5-6 times. Furthermore, the response was rapid as the mRNA levels increased approximately 3.5 times after 1 d of the cysteine infusion and, by 1 d post-infusion, they had fallen, approaching their basal level. No changes in the mRNA levels encoding the intermediate filament or the other keratin-associated protein families of lower cysteine content were observed. Concomitantly, two-dimensional polyacrylamide gel electrophoresis analysis of wool proteins showed a striking increase in the abundance of a group of cysteine-rich keratin-associated proteins in the wool by the end of the infusion period, returning to basal levels by 3 weeks later. At the cellular level, KAP4 expression was localized to the follicle paracortical cells, and the proportion of paracortical cells and the extent of KAP4 expression paralleled the changes in the cysteine infusion status of the sheep.

Transgenic sheep and wool growth: possibilities and current status.

Merino wool is the result of generations of selection, yet improvements in wool quality and performance are still being sought. Through gene manipulation, sheep transgenesis offers possibilities of understanding the relationship between wool keratin protein composition and fibre structure and properties and of introducing novel changes to fibre properties and growth rates. We have established an efficient sheep transgenesis programme with an overall transgenic rate of 2.1% of zygotes injected. However, by incorporating in vitro culture and assessment of injected zygotes, this equates to a transgenic rate of 13% from 516 lambs born. With the first keratin gene construct, a wool keratin type II intermediate filament gene, four live F0 transgenic sheep have been produced and all express the transgene. In one of them, the highest expressor, phenotypic and ultrastructural changes were evident in the fleece. To improve wool growth rate by increasing the supply of cysteine to the follicle, transgenic sheep are being produced carrying the two genes necessary for endogenous cysteine synthesis. Three promoters have been tested driving the cysteine synthesis genes: two general promoters, the Rous sarcoma virus long terminal repeat and mouse phosphoglycerate kinase promoter, and a rumen-specific promoter from the sheep small proline-rich protein gene. To date, one transgenic sheep (bearing the small proline-rich protein promoter constructs) has produced cysteine in the rumen, although the amount was low at 3 months of age and not detectable at 6 months.

Organization and expression of hair follicle genes.

Several families of proteins are expressed in the growth of hair and an estimated 50-100 proteins constitute the final hair fiber. The cumbersome nomenclature for naming these different proteins has led to a proposal to modify that which is currently used for epidermal keratins. Investigations of the organization of hair genes indicate that the members of each family are clustered in the genome and their expression could be under some general control. Interestingly, the protein called trichohyalin, markedly distinct from the hair proteins, is produced in the inner root sheath cells and the gene for it has been found to be located at the same human chromosome locus as the genes for profilaggrin, involucrin, and loricrin. A mainstream objective is to identify controls responsible for the production in the hair cortex of keratin intermediate filaments (IFs) and two large groups of keratin-associated proteins (KAPs) rich in the amino acids cysteine or glycine/tyrosine. A specific family of cysteine-rich proteins is expressed in the hair cuticle. Comparisons of promoter regions of IF genes and KAP genes, including a recently characterized gene for a glycine/tyrosine-rich protein, have revealed putative hair-specific motifs in addition to known elements that regulate gene expression. In the sheep, the patterns of expression in hair differentiation are particularly interesting insofar as there are distinct segments of para- and orthocortical type cells that have significantly different pathways of expression. The testing of candidate hair-specific regulatory sequences by mouse transgenesis has produced several interesting hair phenotypes. Transgenic sheep over-expressing keratin genes but showing no hair growth change have been obtained and compared with the equivalent transgenic hair-loss mice. Studies of the effects of amino acid supply on the rate of hair growth have demonstrated that with cysteine supplementation of sheep a perturbation occurs in which there is a markedly increased level of only one type of mRNA and the ration of para- to orthocortical cells is increased. A molecular explanation of this phenomenon is being sought.

Analysis of the sheep trichohyalin gene: potential structural and calcium-binding roles of trichohyalin in the hair follicle.

Trichohyalin is a structural protein that is produced and retained in the cells of the inner root sheath and medulla of the hair follicle. The gene for sheep trichohyalin has been purified and the complete amino acid sequence of trichohyalin determined in an attempt to increase the understanding of the structure and function of this protein in the filamentous network of the hardened inner root sheath cells. Sheep trichohyalin has a molecular weight of 201,172 and is characterized by the presence of a high proportion of glutamate, arginine, glutamine, and leucine residues, together totaling more than 75% of the amino acids. Over 65% of trichohyalin consists of two sets of tandem peptide repeats which are based on two different consensus sequences. Trichohyalin is predicted to form an elongated alpha-helical rod structure but does not contain the sequences required for the formation of intermediate filaments. The amino terminus of trichohyalin contains two EF hand calcium-binding domains indicating that trichohyalin plays more than a structural role within the hair follicle. In situ hybridization studies have shown that trichohyalin is expressed in the epithelia of the tongue, hoof, and rumen as well as in the inner root sheath and medulla of the hair follicle.

Sequence, expression, and evolutionary conservation of a gene encoding a glycine/tyrosine-rich keratin-associated protein of hair.

In hair differentiation several families of keratin proteins with distinctive amino acid compositions are produced. To study the role and regulation of one of these families, the glycine/tyrosine-rich keratin-associated proteins encoded by the KAP6 gene family, a partial wool follicle cDNA clone encoding a sheep KAP6 protein was sequenced and the corresponding gene isolated from a sheep cosmid library. The KAP6.1 gene encodes a basic protein of 82 amino acids (M(r) = 8,296) with a combined glycine and tyrosine content of approximately 60 mol%. There are several KAP6 genes in the sheep genome, all located within a 1,050-kilobase SfiI fragment. Northern blot analysis demonstrated that at least one member of the KAP6 family is expressed in the wool follicle. A rabbit KAP6 gene was isolated and its sequence and expression patterns were compared with the sheep gene. The sheep and rabbit genes have a nucleotide sequence identity of 89%, suggesting that they are equivalent genes and indicating strong selection pressure during evolution. Both genes contain several conserved sequence motifs of 7-9 nucleotides in their 5'-flanking regions that may be involved in the regulation of their expression. Localization of KAP6 mRNAs in sheep wool and rabbit hair follicles by in situ hybridization suggests that the genes are expressed in the cells of the hair shaft cortex in varying expression patterns. KAP6 expression starts relatively late in hair follicle differentiation, and the proportion of hair cortical cells that express it may change from follicle to follicle.

Complete sequence of a hair-like intermediate filament type II keratin gene.

The Intermediate Filament (IF) superfamily comprises several multigene families, of which the two keratin families are the largest. The keratin IF genes are expressed in epithelial tissues in differentiation-specific patterns and recently we reported the sequence and expression of a hair IF type II keratin gene (KRT2.9). Two related genes were present in the cosmid containing KRT2.9 and we have now sequenced one of them and found that it encodes a hair-like IF type II protein (KRT2.13). However, KRT2.13 is not expressed in the hair follicle. Interestingly there is significant sequence homology between introns 1, 5 and 6 of KRT2.13 and KRT2.9 to suggest gene conversion of these regions or possibly conservation of functional sequences.

Mapping of the trichohyalin gene: co-localization with the profilaggrin, involucrin, and loricrin genes.

The chromosomal location of the gene encoding the human hair follicle protein trichohyalin has been determined by in situ hybridization. The human gene has been localized to the region 1q21.1-1q23 (probably 1q21.3) using a sheep trichohyalin cDNA probe. The genes encoding three other epithelial proteins, namely, profilaggrin, involucrin, and loricrin, are also located in the same region of chromosome 1, which, together with their similar gene and protein structures, suggests that the four proteins form a novel superfamily of epithelial structural proteins.

Coexpression of the cys E and cys M genes of Salmonella typhimurium in mammalian cells: a step towards establishing cysteine biosynthesis in sheep by transgenesis.

The Salmonella typhimurium genes for serine acetyltransferase (cys E) and O-acetylserine sulphydrylase B (cys M) were isolated and characterized in order to express these as transgenes in sheep to establish a cysteine biosynthesis pathway and, thereby, to achieve an increased rate of wool growth. Comparison of the S. typhimurium and Escherichia coli genes showed considerable homology, both at the nucleotide and amino acid sequence levels. The in vitro and in vivo expression studies showed that both genes could be transcribed and translated in eukaryotic cells and that their products could function as active enzymes. The cys M gene of S. typhimurium possessed a GUG initiation codon, like its E. coli counterpart, but translation could be initiated using this codon in eukaryotic cells to give an active enzyme product. Chinese hamster ovary cells, stably transfected with a tandem arrangement of the two genes, showed a capacity to synthesize cysteine in vivo, indicating the establishment of a cysteine biosynthesis pathway in these cells. The measured levels of activity of the gene products suggest that improved wool growth is possible by transgenesis of sheep with these genes.

Localization by in situ hybridization of a type I keratin intermediate filament gene (Krt-1.14) to band D of mouse chromosome 11.

A probe from the 3' noncoding region of a murine type I keratin intermediate filament (IF) gene (Krt-1.14) localizes to band D of murine Chromosome 11 using in situ hybridization. This localization provides a physical confirmation of the assignment of the type I keratin genes by linkage analysis in the mouse. It also demonstrates that the Krt-1.14 genes are at a single locality in the mouse in contrast to the two locations on the short and long arms of chromosome 17 in humans.

Regulation of keratin gene expression in hair follicle differentiation.

In hair growth, as the follicle bulb cells rapidly differentiate into either cortical or cuticle hair keratinocytes, about 50-100 keratin genes are transcriptionally activated. However, this complexity can be reduced to several, highly conserved gene families. In studying the regulation of keratin gene expression in the hair follicle we have isolated genes from most of these families and have examined their expression patterns by in situ hybridization. In the cortical keratinocytes striking patterns of keratin gene expression exist, suggesting that different transcriptional hierarchies operate in the various cell types. Comparisons of the keratin gene promoter regions indicates conserved sequence motifs that could be involved in determining these cell specificities. Similarly, we have isolated related sheep and human cuticle keratin genes and find conserved DNA motifs and expression patterns in cuticle cell differentiation. Additionally, the expression of sheep wool follicle IF and high-sulfur keratin genes in transgenic mice suggests that the regulatory DNA elements and proteins of hair keratin genes are functionally conserved between mammals.

The structure and expression of a gene encoding chick claw keratin.

A cDNA library was constructed from embryonic chick claw mRNA and a claw keratin (cKer)-encoding clone was isolated and sequenced. Subsequently, a genomic clone, containing four cKer-encoding genes (cKer) was isolated and one of the genes (cKer1) was completely sequenced. The cKerl gene appears to be differentially expressed in the keratinizing tissue appendages of the embryonic chick, being abundantly expressed in the claw and at a low level in feather tissue. Comparison of the deduced amino acid (aa) sequence of the cKer to those of feather (fKer) and scale keratins (sKer) showed that the regions conserved between fKer and sKer are also found in the cKer. The glycine-rich as repeat region characteristic of sKer is also present in a shortened form in the cKer sequence. Like the fKer genes (fKer) and the feather histidine-rich protein-encoding gene (HRP), the cKer1 gene also contains one intron which interrupts the 5'-noncoding region at an equivalent position to that found in the fKer and HRP genes. Genomic Southern analysis using the cKer cDNA as a probe indicated the presence of several related genes in the chick genome.

An ultrahigh-sulphur keratin gene of the human hair cuticle is located at 11q13 and cross-hybridizes with sequences at 11p15.

A human hair cuticle ultrahigh-sulphur keratin Q (UHSK) gene (KRN1) has been mapped by Southern analysis of a somatic cell hybrid panel and by in situ hybridization. A probe containing the coding region of this gene mapped to 11pter- greater than 11q21 using the hybrid cell panel and on in situ hybridization mapped to two regions on chromosome 11: the distal part of 11p15, most likely 11p15.5, and the distal part of 11q13, most likely 11q13.5. A probe from the 3' noncoding region of KRN1 mapped to 11q13.5 indicating that this was the map location of the cloned gene. The sequence of 11p15.5 is termed KRN1-like (KRN1L). The results reveal that the cuticle UHSK gene family is clustered in the human genome.