AIMS:To investigate the location of keratin-associated proteins (KAPs) in developing hair fiber cuticle cells using transmission electron microscopy with immunogold techniques and specific antibodies. Other studies were aimed at detecting the presence of cornified envelope proteins including involucrin and loricrin. MATEIALS AND METHODS: Polyclonal antibodies were produced in rabbits against peptides from KAPS 5.1, KAPS 10.1 ultra high-sulfur proteins.RESULTS:The KAP proteins were found to form part of the developing exocuticle and a- layer. Cornified envelope proteins (involucrin and loricrin) were absent consistent with recent findings.CONCLUSIONS:The results have been discussed in terms of a revised model for fiber cuticle surface barriers including their role in fiber cuticle surface function.

Ten years ago, Hardy (1992) wrote a timely review on the major features of hair follicle development and hair growth which she referred to as a secret life. Many of these secrets are now being revealed. The information discussed in this brief review comprises the structure of the hair and hair follicle, the continuing characterisation of the genes for keratin and keratin associated proteins, the determination of the location of their expression in the different cell layers of the hair follicle, molecular signals which control keratin gene expression and post-translational events in the terminal stages of hair formation.

The intermediate filaments (IF) in trichocyte (hard-α) keratin form ordered aggregates that are infiltrated by sulfur-rich and tyrosine-rich proteins during fiber development to give a filament-matrix texture, which is stabilized in the later stages by the formation of disulfide linkages. Two polymorphic forms of macrofibril are found in the cortical cells of fine Merino wool. In the first the packing of the IF in the macrofibril is quasi-hexagonal whilst in the second the IF are packed in cylindrical sheets around a central core. In hairs the second type generally predominate. In the present contribution specific models for the mechanisms of nucleation and growth are developed for the two types of macrofibril and their applicability tested by analyzing transmission electron micrographs of wool and hair. Evidence is presented which supports the idea that sheet formation plays an important role in both types of macrofibril assembly and it is suggested that differing intersheet interactions are responsible for the differences between the ortho- and para-types. It is shown that the increase in IF tilt with radius in the ortho-type can be related to the surface lattice of the IF as determined from X-ray diffraction studies. Two possible types of intersheet interaction in the ortho-type are discussed, the first leading to an increase of around 0.5° in IF tilt per layer and the second leading to a much larger tilt of 9.4° per layer. A crude estimate based on the decrease in visibility of the IF with increasing radius in cross-section yielded a value of 0.35°–0.7°.

The development of new synthetic materials used in the manufacture of textile fibres has greatly expanded the range of properties able to be delivered by apparel and other weaves and has also exceeded the capabilities of natural fibres such as wool and cotton in these uses. Consequently, producers of natural fibres have sought to improve fibre properties and broaden their property range. For wool, a relatively coarse and weak fibre, this has led to selection for animals which synthesise finer fibres for apparel fabrics and among other features, those which produce highly crimped fibres to improve wool "bulk". In addition, post-farm treatments of the fibre and its surface have recently been used to create wool fibres more suitable for general and sport apparel manufacture. For example, pure wool or blended-wool garments with "easy care" features and with better moisture conduction qualities have recently been produced. Such products provide solutions for the present but are costly to manufacture. If similar properties could be engineered into wool through manipulation of the chemical composition of fibres on-farm, during wool growth, considerable savings in off-farm energy consumption and posttreatment processing costs could be achieved. Transgenesis provides an opportunity to develop wools with different protein compositions and properties that could be an advantage during processing or which might provide novel qualities to the end product. Here we describe experiments to create wool fibres with improved qualities; sheep transgenesis using wool fibre keratin and keratin-associated protein genes mis-expressed in the wool follicle cortex.

Genetic engineering methods are being investigated for their potential to produce new kinds of textile fibres. The systems fall into two main groups. There are those systems that can produce monomeric protein molecules in solution from appropriately engineered genes and include expression in bacteria, cell cultures or in the milk of transgenic animals such as goats or sheep. The protein monomers are then isolated from the chosen system and spun and drawn into fibres. The other approach is to modify keratin fibres such as wool by expressing other proteins in the internal components by transgenesis. These systems are briefly reviewed with a discussion of the properties being sought and the progress to date including some changes to cellulose fibres.

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 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.

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.

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.

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.

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.

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.

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 (cKerl) 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 aa 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 cKerl 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.

Annals of the New York Academy of SciencesVolume 642, Issue 1 p. 64-80 Trichohyalin and Matrix Proteinsa GEORGE E. ROGERS, GEORGE E. ROGERS Department of Biochemistry University of Adelaide South Australia 5000, AustraliaSearch for more papers by this authorMICHAEL J. FIETZ, MICHAEL J. FIETZ Department of Biochemistry University of Adelaide South Australia 5000, AustraliaSearch for more papers by this authorANTONIO FRATINI, ANTONIO FRATINI Department of Biochemistry University of Adelaide South Australia 5000, AustraliaSearch for more papers by this author GEORGE E. ROGERS, GEORGE E. ROGERS Department of Biochemistry University of Adelaide South Australia 5000, AustraliaSearch for more papers by this authorMICHAEL J. FIETZ, MICHAEL J. FIETZ Department of Biochemistry University of Adelaide South Australia 5000, AustraliaSearch for more papers by this authorANTONIO FRATINI, ANTONIO FRATINI Department of Biochemistry University of Adelaide South Australia 5000, AustraliaSearch for more papers by this author First published: December 1991 https://doi.org/10.1111/j.1749-6632.1991.tb24381.xCitations: 18 a This work was supported by grants from the Australian Research Council and from the Wool Research and Development Council on the recommendation of the Australian Wool Corporation. AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume642, Issue1The Molecular and Structural Biology of HairDecember 1991Pages 64-80 RelatedInformation

We have produced transgenic mice containing up to 250 copies of a sheep wool intermediate filament keratin gene to study the effect of its expression on hair structure and development. Several transgenic lines expressed the gene and in the one containing 250 transgenes, a pattern of hair‐loss and regrowth was stably established. Successive waves of hair growth follow periods of denuding like the natural progression of hairs in the mouse hair cycle. By in situ hybridization we have shown that the sheep transgenes are expressed at the correct stage in mouse hair development and at a high level. The transgenic hairs contain not only an elevated level of intermediate filament keratin protein but also a decreased level of the filament‐associated proteins. This imbalance disrupts the normal ordered array of these proteins in the cells of the hair cortex and leads to weakened fibres which are prematurely lost.

Microinjected sheep zygotes were cultured in synthetic oviduct fluid medium (SOFM) for either 1 or 3 days and their subsequent developmental capacity was compared with that of microinjected zygotes cultured in vivo. Two experiments were carried out, using zygotes microinjected with one of three gene constructs containing the CysE and CysM genes from Salmonella typhimurium. In Experiment 1, microinjected zygotes were allocated to one of three treatments: (1) immediate transfer to recipient ewes (in vivo culture) followed by recollection 1 or 3 days later and subsequent transfer of viable embryos to other recipient ewes, (2) culture in SOFM (in vitro culture) for either 1 or 3 days before transfer to recipient ewes, and (3) immediate transfer to recipient ewes without subsequent interference. Recipient ewes were slaughtered on Day 14 of pregnancy and the number of elongated conceptuses determined. Although fewer zygotes failed to divide during in vitro culture than during in vivo culture, there were, overall, no significant differences between treatments in the percentage of zygotes that developed into elongated conceptuses (32.6-50.0%). In Experiment 2, microinjected zygotes were transferred immediately to recipient ewes or cultured in vitro for either 1 or 3 days before transfer. The number of fetuses per ewe on Day 50 of pregnancy and the number of lambs delivered per ewe were recorded. Neither the percentage of recipient ewes that became pregnant (overall 114/166, 68.7%) nor the percentage of zygotes that developed into lambs (overall 186/803, 23.2%) was significantly influenced by the culture treatment or by the gene construct microinjected.(ABSTRACT TRUNCATED AT 250 WORDS)

Trichohyalin is a highly expressed protein within the inner root sheath of hair follicles and is similar, or identical, to a protein present in the hair medulla. In situ hybridization studies have shown that trichohyalin is a very early differentiation marker in both tissues and that in each case the trichohyalin mRNA is expressed from the same single copy gene. A partial cDNA clone for sheep trichohyalin has been isolated and represents approximately 40% of the full-length trichohyalin mRNA. The carboxy-terminal 458 amino acids of trichohyalin are encoded, and the first 429 amino acids consist of full- or partial-length tandem repeats of a 23 amino acid sequence. These repeats are characterized by a high proportion of charged amino acids. Secondary structure analyses predict that the majority of the encoded protein could form alpha-helical structures that might form filamentous aggregates of intermediate filament dimensions, even though the heptad motif obligatory for the intermediate filament structure itself is absent. The alternative structural role of trichohyalin could be as an intermediate filament-associated protein, as proposed from other evidence.

The major histological components of the hair follicle are the hair cortex and cuticle. The hair cuticle cells encase and protect the cortex and undergo a different developmental program to that of the cortex. We report the molecular characterization of a set of evolutionarily conserved hair genes which are transcribed in the hair cuticle late in follicle development. Two genes were isolated and characterized, one expressed in the human follicle and one in the sheep follicle. Each gene encodes a small protein of 16 kD, containing greater than 50 cysteine residues, ranging from 31 to 36 mol% cysteine. Their high cysteine content and in vitro expression data identify them as ultra-high-sulfur (UHS) keratin proteins. The predicted proteins are composed almost entirely of cysteine-rich and glycine-rich repeats. Genomic blots reveal that the UHS keratin proteins are encoded by related multigene families in both the human and sheep genomes. Tissue in situ hybridization demonstrates that the expression of both genes is localized to the hair fiber cuticle and occurs at a late stage in fiber morphogenesis.