Biochimica et Biophysica Acta 824 (1985) 201-208 201 Elsevier

BBA 91437

I s o l a t i o n a n d c h a r a c t e r i s a t i o n o f k e r a t i n m R N A f r o m th e sca l e e p i d e r m i s o f t h e

e m b r y o n i c c h i c k

S.D. W i l t o n , L .A . C r o c k e r a n d G . E . R o g e r s

Department of Biochemistry, University of Adelaide, Adelaide, South Australia, 5000 (Australia)

(Received July 31st, 1984) (Revised manuscript received December 17th, 1984)

Key words: Keratin; mRNA isolation; Cell free translation; Nucleic acid sequence; Cloning; (Chicken)

The keratin polypeptides of the epidermis from the leg scale region of 17-day-old embryonic chicks were extracted as S-carboxymethylated derivatives and characterised by electrophoresis on SDS and pH 9.5 urea gels including a combination of both in two dimensions. Proteins were isolated that gave X-ray diffraction patterns typical of a- and ~- (avian feather) keratins. An mRNA fraction was isolated from 17-day-old scale tissue by guanidinium chloride extraction and sucrose gradient fractionation. The mRNA was translated in the wheat germ system to give a major product indistinguishable from the molecular weight class ( M r 14 500) of scale ~-keratin polypeptides. A cDNA library was constructed in pBR322 from a 15 S mRNA subfraction and two recombinant clones were selected by their strong hybridisation to cDNA prepared from the 15 S mRNA. The sequencing of these has yielded details of the relatedness of two scale keratin genes including their 3' untranslated regions. Almost half of the protein sequences of the two homologous scale keratins has been deduced and a notable feature of the scale keratin structure appears to be the presence of at least two sequence domains consisting of 13 amino acid repeats.

Introduction

Avian feathers and scales are believed to have evolved from the same ancestral scale tissue [1]. Similarities between feather and scale proteins in the amino acid sequences of their respective amino and carboxyl terminii, a polar distribution of half-cystine residues and an internal hydrophobic region indicate that the same ancestral gene was selected for amplification and divergence [2]. While the avian feather keratin system has been studied in considerable detail at both the protein level [3-6] and at the nucleic acid level [7-9] only limited studies at the protein level of the avian scale keratin system have been undertaken [2]. Detailed comparisons between these families of proteins, including their divergence from the ancestral scale gene can only be achieved by ex-

amination of the nucleotide sequences. The initial aims of the present study were first

to identify the major keratin protein components of scale epidermal tissue and, in turn, to obtain identifiable scale keratin mRNA from which pure mRNA species could be isolated through the use of recombinant DNA methods. Since a minimum of nine keratin genes, each coding for a closely related protein, are expressed in scale tissue [2], the isolation of a single polypeptide for protein sequencing is very difficult. Thus, the only possible approach to determine unambiguously the primary structure of a scale keratin protein is via the nucleotide sequence of its corresponding mRNA. Furthermore, once such a nucleotide sequence has been isolated and identified, it becomes possible to study the organization and arrangement of scale keratin genes in the genome and to allow a direct

202

comparison to be made between the protein cod- ing and regulatory regions of the feather and scale keratin genes. The present paper describes the isolation of pure cDNAs coding for scale keratins and the comparison of these sequences of the scale keratin family.

Methods

Source and preparation of keratins Scale tissue from embryonic (17-day-old) chick-

ens (White Leghorn, strain Para 3 obtained from Parafield Poultry Station, Parafield, South Austra- lia) was taken from the shank region and im- mersed in 20% sodium bromide for 1 h at 23°C in order to separate the epidermis from the underly- ing dermis. Epidermal proteins were extracted and S-carboxymethylated as described by Kemp and Rogers [4].

Polyacrylamide gel electrophoresis Polyacrylamide gel electrophoresis at pH 9.5

was carried out essentially as described [4], except that ammonium persulphate and N,N,N',N'-tetra- methylethylenediamine (TEMED) were used to polymerize the gel instead of riboflavin. SDS-poly- acrylamide gel electrophoresis was carried out using the procedure of Weeds [10] with the ad- dition of 0.1% SDS to the reservoir buffer.

Scale RNA extraction and fractionation Skin (epidermis and dermis) from the shank of

17-day-old embryonic chicks was used for the extraction of RNA essentially as described by Brooker et al. [11] except that the ethanol precipi- tation conditions were adjusted to include smaller RNA species. Very briefly, scale tissue was ho- mogenized in a solution containing guanidine hy- drochloride. After the addition of ethanol, the insoluble material was collected, resuspended in another guanidine hydrochloride solution and ethanol precipitated once more. The insoluble fraction was resuspended in 7 M urea then phe- nol /ch loroform extracted twice and ethanol pre- cipitated. Total scale RNA was loaded on to linear 10-40% sucrose gradients, prepared by the freeze- thaw method of Davis and Pearson [12] and centrifuged for 15 h at 37000 rpm at 4°C. When

denaturing conditions were required, total RNA was fractionated on agarose gels containing meth- ylmercuric hydroxide [13]. The RNA was electro- eluted from gel slices.

Cell-free protein synthesis The in vitro translation assay using wheat germ

$30 was as described [14]. RNA was incubated with the wheat germ $30 fraction in the presence of [3H]leucine and total translational activity was quantitated by measuring the incorporation of ra- dioactive amino acid into trichloroacetic acid-pre- cipitable material [15]. The translation products were S-carboxymethylated [4] and then identified using the previously described polyacrylamide gel systems and fluorography [16].

Preparation of double-stranded (ds) eDNA The procedure used for the preparation of ds-

cDNA for cloning is described below and is a modification of the procedure described by Rou- geon and Mach [17].

(a) First strand synthesis. Oligo(dT) primed re- verse transcription of the 15 S RNA fraction ob- tained in the sucrose gradient fractionation was carried out in a 125 ~1 reaction mix containing 10 ~g RNA, 480/~M each of dATP, dTTP and dCTP, 0.06 /~M [c~-32p]dGTP, 72 /~M dGTP, 10 mM Tris-HC1 (pH 8.3), 8 mM MgC1 z, 10 mM di- thiothreitol and 10/~g/ml oligo(dT). 100 units of AMV reverse transcriptase (obtained from Dr. J. Beard) was added, and the mixture was incubated at 42°C for 40 min.

(b) Synthesis of the second strand. Following the synthesis of the first strand, the reaction mixture was heated in a boiling water bath for 2 min and snap chilled on ice, and a further 0.25 mM final concentration of deoxynucleotide triphosphates and 1.0 mM dithiothreitol were added. 40 units of reverse transcriptase were added and incubated for 5 h at 37°C. A further 40 units of enzyme were added after 2.5 h. The ds-cDNA was treated with S1 nuclease, phenol extracted and passed through a Sephadex G-50 column (0.8 × 33 cm) to remove unincorporated nucleotides. The material was then centrifuged through 5-20% sucrose gradients (36000 rpm, 15 h, 4°C) to select double-stranded transcripts in excess of 500 basepairs.

Homopolymer tailing 1 ttg of PstI digested pBR322 was incubated in

the presence of 1 nmol (26 C i / nm o l ) [3H]dGTP, 10 mM sodium cacodylate (pH 7.5), 0.1 mM di- thiothreitol, 4 mM MgCI2 and 5 units calf thymus terminal transferase (Ratliffe Biochemicals). Dou- ble-stranded cDNA was incubated under similar conditions using [3H]dCTP and CoC12 in place of [3H]dGTP and MgC12. The reaction at 4°C was followed by the conversion of the [3H]deoxynuc- leotides into a trichloroacetic acid insoluble form. When an average of 20-25 nucleotides per end had been added, the reaction was terminated by the addition of an equal volume of 20 mM EDTA.

Annealing reaction The dG-tailed plasmid DNA and dC-tailed ds-

c D N A were mixed in equimolar amounts in 10 mM Tris (pH 7.5), 1 mM EDTA and 200 mM NaC1 and heated to 60°C for 10 min. The mixture was then incubated at 45°C for 60 min and al- lowed to gradually cool to 4°C overnight.

Transformation and screening for recombinants A 50 ml culture of E. coli ED8654 (rK-mK+sup E

sup v trp R) was grown in L broth (1% Bactotryp- tone, 0.5% yeast extract, 1% NaC1) to a density of 0.5 A600. The cells were made competent and transformed essentially as described [18], except that the final incubation in 100 mM CaC12 was 4 h at 4°C. The transformed cells were plated out on L-broth agar plates containing tetracycline at a final concentration of 20 /~g/ml. The resulting colonies were screened for their resistance to tetra- cycline and ampicillin by replica plating on to L-broth agar plates containing either tetracycline (20 /~g/ml) or ampicillin (30 /~g/ml). Colony hybridization was carried out by the method of Grunstein and Hogness [19].

Isolation of plasmid DNA Plasmid D N A was prepared essentially as de-

scribed [20].

DNA sequencing reactions D N A fragments to be sequenced were either

labelled at the 5' ends by polynucleotide kinase [21] or, more commonly, at the 3' ends by 'end filling' with the large fragment of D N A Poll

203

(Klenow). Chemical reactions specific for G, G + A, T + C, C and T were carried out as described [21,22]. Samples were dissolved in a formamide loading solution, heat-denatured and fractionated on polyacrylamide sequencing gels, 0.5 mm thick.

Results

Characterization of S-carboxymethylated scale keratins

Gel electrophoresis of S-carboxymethylated scale keratins at pH 9.5 gave a band pattern (Fig. l a) which was similar to that already reported [4]. SDS gel electrophoresis of these proteins divided the scale keratins into two main molecular-weight classes (Fig. lb). Those of least electrophoretic

a. ES A S

As

Bs

b. ES A S

!

~SK I i i !

i ~=~ i ̧¸̧ ̧

/~SK

Fp ~ i ¸̧ ̧ /!

+ -4- Fig. 1. Fractionation of S-carboxymethylated keratins from embryonic (ES) and adult (AS) scale tissue on (a) pH 9.5 urea and (b) SDS-polyacrylamide gel systems. The band notation is from Dhouailly et al. [23], thus A s, B s refer to minor and major scale keratin groups respectively, ask and flsK are, respectively, the high-molecular-weight a-type scale keratins and main scale keratin bands. Fp is the fast protein component which has a higher mobility than the scale keratins in SDS gels.

204

mobility, corresponding to a molecular weight of between 50000 to 60000, were shown by X-ray diffraction studies to contain some a-helix (Mac- Rae, T.P., personal communication) indicative of the presence of an a-conformation. The major class of scale proteins with a molecular weight of 14500 [2] gave X-ray reflections (McRae, T.P., personal communication) indicative of the pres- ence of a fl-conformation.

To clarify the nomenclature based on the SDS and pH 9.5 urea gel systems, two-dimensional electrophoresis was carried out on total scale pro- teins. The two-dimensional separation of scale components (Fig. 2) demonstrated that the A~ and B S groups from the high-pH gel separation, as designated by Dhouailly et al. [23] correspond to the a-type and fl-type keratins, respectively. The B-type keratins appeared to be a complex class of proteins, and at least 12 individual components were distinguished in the riSK region shown in Fig. 2.

v = ~ S K

i il

Fp ÷

F i r s t D i m e n s i o n ( pH 9"5)

A s Bs I I I "1

Fig. 2. Two-dimensional fractionation of S-carboxymethylated keratins from embryonic scale tissue. Fractionation in the first dimension took place in a pH 9.5 urea tube gel which was subsequently embedded in an SDS-polyacrylamide slab gel for electrophoresis in the second dimension. (A second pH 9.5 urea tube gel which had been fixed and stained with Coomassie blue is shown on top of the slab gel for reference). Shortly before termination of the separation in the first dimension, a second loading of embryonic scale keratin was carried out and allowed to just enter the gel to provide markers for the second dimen- sion. Several electrophoretically distinct scale a- and fl-keratins can be discerned although the bands vary considerably in relative intensity. This fractionation demonstrates that the A s and B s groups of the pH 9.5 urea single-dimension gel, corre- spond to the aSK and flSK classes of the SDS-gel, respectively.

Scale fl-keratin rnRNA purification Sucrose gradient fractionation of total RNA

from 17-day-old embryonic scale tissue and the subsequent refractionation of 6-17 S RNA on 10-40% sucrose gradients are shown in Fig. 3a and b, respectively. Each fraction was assayed for scale B-keratin mRNA activity in the wheat germ cell-free translation system. Identification of the scale fl-keratin mRNA was based upon the elec- trophoretic properties of the translation products in the SDS and pH 9.5 urea polyacrylamide gel systems (Fig. 4a and b).

From the number of electrophoretically distinct translation products, it appeared that the RNA was a mixture of mRNAs distributed over the range 9-18 S. It must be pointed out that the cell-free wheat germ translation conditions had been adjusted to optimise the expression of scale /~-keratin mRNA. The optimal concentrations of K + for the translation of feather and scale fl-kera- tin m R N A was found to differ markedly, i.e., 70 and 40 mM, respectively (results not shown). In-

a b

l 165

Sed imen ta t i on Sed imen t a t~or , J

Fig. 3. (a) Sucrose gradient sedimentation of total RNA ex- tracted from embryonic scale tissue. (b) Recentrifugation of pooled 6-17 S RNA. The 6-17 S RNA from five gradients shown in (a) were pooled, recovered by ethanol precipitation, dissolved in 0.5 ml 100 mM Tris-HCl (pH 9.0), 0.1% SDS and layered over 10-40% (w/v) sucrose gradients. The 9.5, 12, 15 and 16.5-18 S peaks were isolated as shown.

205

a WG 9.5S 12S 15S 16.5S USK b USK WG 9.5S 12S 15S 16.5S

As

1 ~'SK

I -?SK

-Fp

Bs

+ +

i !i~ I i~i!ii ~!~i~i~ili~! '~!~ i~i~ ~

• i i!~ii!i ~!i~ii!iii~i~ ~ ~i ~ ?i~ii~ ~ ~!~i~

I i !i iiiii!i!iiiiiii ii

Fig. 4. Fractionation of S-carboxymethylated translation products from 9.5, 12, 15 and 16.5-18 S RNA fractions from embryonic scale tissue on (a) SDS-polyacrylamide and (b) pH 9.5 urea polyacrylamide gel systems. WG, endogenous wheat germ activity; USK, 14C-labelled total scale keratins. The protein groups A s, B s, asK, /3sK are as described in Fig. 1.

deed, it may have been the low K + concentrations which did not allow the efficient expression of many other cellular mRNAs, which would have been present in a lower abundance than the scale fl-keratin mRNA. The Mg 2+ concentration used was 3 mM in both cases. Significant amounts of translationally active scale fl-keratin m R N A were found in the 12 S and the 15 S RNA fractions. These fractions also contained other mRNAs, which directed the cell-free synthesis of a number of polypeptides of lower molecular weights than those of the scale fl-keratins (Fig. 4a). The origin and nature of these smaller polypeptides has not been determined. It was of interest that only a single peak of translational activity was observed after total RNA from scale tissue had been frac- tionated on a methylmercuric hydroxide agarose gel instead of a non-denaturing sucrose gradient

and the RNA fractions eluted and translated in the wheat germ cell-free system (Fig. 5a). The single peak of activity corresponded to an m R N A of between 800 and 1000 bases. The translation product of this peak was identified as scale fl- keratin (Fig. 5b).

Sequences of scale fl-keratin recombinant plasmids Recombinants (Tet res and AMP . . . . ) were ob-

tained from the cloning of the ds cDNA prepared from the 15 S m R N A subfraction described above. From these, 65 recombinants were further screened for B-keratin nucleotide sequences with a [32p]cDNA probe prepared from the 15 S subfrac- tion of scale RNA. Approximately half of these recombinants were found to hybridize to the scale keratin cDNA probe, and several of them were chosen for further study. Finally, on the basis of

206

a.

24

20

3 H - L e u 18

incorporated cpm x 10-4 1 2

18S 12S 5S

2 3 4 5 6 7 8 910111213141516171819202122232425

Slice Number 18S ~ 5 S

Slice N u m b e r BSK 10 11 12 13 14 15 W G

Fig. 5. (a) Distribution of mRNA activity after total RNA from embryonic scale tissue was denatured and fractionated on the basis of size. RNA was electroeluted from 2 mm thick gel slices after electrophoresis through a 1% agarose gel containing 4 mM CH3HgOH and translated in the wheat germ cell-free system. (b) Fluorograph of the SDS-polyacrylamide gel electrophoresis profile of the S-carboxymethylated translation products from gel slices 10-15 which represented the peak of mRNA activity in (a). These translation products co-migrated with the ~4C scale fl-keratin marker flsK. The wheat germ control, WG, had no RNA added.

the autoradiographic intensity after hybridization with the probe, two clones, pCSK9 and 12, were selected for sequencing. It should be noted that in both cases, the cloned D N A could not be com- pletely resected with PstI, and this was subse- quently found by sequence analysis to have been due to deletions occurring around the PstI cloning site of pBR322 prior to the addition of the homo- polymer tails. Presumably, the deletions arose from exonuclease contamination of the PstI endo- nuclease used in the preparation of the vector. The sequencing strategies employed in determining the nucleotide sequences of pCSK9 and 12 are given in Fig. 6a. Neither pCSK9 or 12 were full-length

5' VECTOR

II Hp S H H

I I I I . I

VECTOR i S S S PHp S H H P H

(3

3' p~CSKg P I ~ IVECTOR

Hpa ]I

Sau96 j

Hinf j

0 ~ K 1 2

VECTOR

Sau96 I

Hpa II

#- - - Psi ]

Hirlf i

pCKS9 GGA GGC TCC ICC CTG GGC IAT GGG GGT CTG TAT GGC TAT GGA GGC TCC TCC CIG gZ~ g ~ ~ . e ~ leu g~y t ~ . gl~ gI~ ~eu t y . ~l~ t y . g~y gi~ ~a. s#~ leu

~y @~ oe~ ee. ~eu gZ~ t,~e gl 9 gZ~ Zeu t 9. gZ~ tg ~y g~y ee~ ee. Zeu

9 GGC TAr GGG GGT CTG TAT GGC CTT -*- GGG AGC TAT GGG GGC TAT GGG GGT CIG g~y ~ . gZ~ ~q teu iv. gZu Zeu g~ ~e. t~. gZp g ,w t~. g~y ~y Zeu

@ly t ~ - gZy gZ 9 Zeu t w - aTu t y ~ ~ ~ ~ ~ ~ y ~ ~ ~ i ~ . ~ i u ~ Z V t ~ . g l ~ . . . . . .

9 TAT GGG GGC TCC CCT GGG TAC AGG GGT CTG IAT GGC TAT GGT AGA TCC TAT GGC

12 . . . . . . . . . . . . . . . . . . . . . . . . . C I T . . . . . . . . . . . . . . . . . . . . . . . gZ~ Zeu ty. Rl~ ~u~ gty a~g se. ~ g~y

9 TCT GGC TAT TGC AGC CCT . . . . . TAC ¢GG TAC AAC AGG TTC CGC CGT GGC AGC

12 --C - - ~ TAC TCC A

a~g ty~ teu ~ gt¥ ed~

9 TGT GGG CCC TGC TAA-342 bases 3' non-coding-ATTAAAGCAATITTCTTCTG-poIyA tel) ey8 gZ~ p.o eg~ t e .~

12 --C -332 bases 3' nOO-COding-ATTAAAAGITTATTGCAFC-polyA taH c~a pZ~ pro ~/~ te .~

b oUm gZy p,.o eyJ

Fig. 6. (a) Sequencing strategies used for the clones pCSK9 and pCSK12. Note that in both cases one of the PstI sites was not regenerated (*) during the cloning procedure (see text). (b) Nucleotide and protein sequences of the two plasmid clones pCSK9 and 12 carrying inserts of scale keratin gene sequence. The complete sequence of pSCK9 is shown, and bases con- served in pCSK12 are indicated by the heavy line. Base changes in the two sequences are shown in the pSCK12 sequence, whereas insertions and/or deletions are indicated by broken lines. Three 39-base repeats are found in pCSK12 (square brackets). Three closely similar repeats are also found in pCSK9 with the additional difference in that the third repeat in pCSK9 is interrupted by a contiguous sequence of 30 extra bases. The short region of 10 amino acids at the C-terminal end of one type of adult scale r-keratin sequenced by Walker and Bridgen [2] is shown for comparison (bold type). In addition, the polyadenylation signals ATTAAA (underlined) and the lengths of the 3' non-coding regions are shown.

copies, since a scale r-keratin gene would be ex- pected to code for about 140 amino acids, whereas the two clones pCSK9 and 12 were found to

207

10 AMINO ACID INSERT

p C S K 9 [ REPEAT 1 I REPEAT 2 [ REPEAT 3 C-TERMINAL eARMO • I COOH

p C S K 1 2 I REPEAT I l R E P E A T 2 I R E P E A T 3 C-TERMINAL ARM ]COOH

Fig. 7. Summary of the partial protein sequences for the two scale keratins derived from plasmid clones pCSK9 and pCSK12 and detailed in Fig. 6a. The diagram is to highlight the three identical repeats (or domains) in pCSK12 and repeats in pCSK9 which are similar except for several amino acid changes (shown by dots) and a ten amino acid sequence inserted in the third repeat (triangle) that divides the repeat into two parts. The C-terminal regions of both pCSK9 and 12 are shown (C-termi- nal arms) and their sequences are conserved, except for three amino acid changes as indicated by the dots on the pCSK9 arm.

contain regions coding for only 73 and 66 amino acids, respectively, equivalent to about half of the scale keratin molecule at the carboxyl end. Only 10 amino acids from the carboxyl end of a scale B-keratin (adult chicken) had been directly se- quenced [2], and there was a close but not identical correspondence with that part of the sequence derived from pCSK9 and 12. The most interesting feature of these nucleotide sequences shown in Fig. 6b is a 39 base sequence repeated exactly three times in pCSK12 and a homologous se- quence repeated twice in pCSK9. In pCSK9 there is a further sequence that has approx. 75% homol- ogy with the others if a 30 base insert is excluded. The block diagrams in Fig. 7 represent the protein sequences derived from pCSK9 and 12 and il- lustrate the relationships between the two proteins with respect to the homologous and repeating 13 amino acid and glycine/tyrosine-rich domains. Differences including the 10 amino acid insert of pCSK9 and single amino acids are shown.

Discussion

Protein components of embryonic chicken scale tis- sue

The keratin proteins of 17-day-old embryonic scale tissue were fractionated into two main groups by their electrophoretic properties. Those in least abundance and with the least mobility in both the

SDS and pH 9.5 gel systems were found by high- angle X-ray diffraction studies to contain a-helix. These a-type keratins have been reported to occur in the hinge regions between the individual scales [24,25]. The predominant class of keratin proteins, however, had a higher mobility than the a-type keratins in the two gel systems used. With a molec- ular weight of about 14500 [2], they were shown by X-ray diffraction studies to contain B-regions typical of the feather keratins.

From the two-dimensional polyacrylamide re- suits presented here, there are at least 12 electro- phoretically distinct scale B-keratin chains in 17- day-old embryonic scale tissue.

The amino acid sequences for scale keratins from cloned cDNA

There were several interesting features of the proteins encoded in pCSK9 and 12. For example, there was not a perfect m~tch between their carboxyl termini and the corresponding region from adult scale (Fig. 6b) published by Walker and Bridgen [2], indicating that different sets of keratin genes are expressed in embryonic and adult scale tissues. Indeed, electrophoretic differences had been demonstrated between embryonic and adult feather keratins [4], but not for the proteins of embryonic and adult scale tissues (results not shown).

The protein sequences shown in Fig. 7 have been divided into three zones such that the two that are highly conserved (greater than 90%) flank a segment where the homology is only about 30%. The mismatched central region may have arisen from a previous recombination event between the nucleotide repeats of related scale keratin genes. Such a sequence which can facilitate genetic ex- change is an obvious evolutionary advantage and may have allowed the vertebrate epidermis to pro- duce a wide variety of keratinized structures such as beak, claw and feather [1].

The combined domains bear considerable simi- larity to the approximately 35-residue sequence containing repeats of Gly-Gly-X (where X can be tyrosine, leucine or phenylalanine) described [2]. This sequence was the major distinguishing feature between feather and scale keratin and has been implicated in the structural role of scale B-keratin [261.

208

The 3' untranslated regions of two scale B-keratin genes

The 3' untranslated regions of pCSK9 and 12 differ markedly in sequence (not shown) and are different in their lengths being, respectively, 362 and 351 bases between their TAA termination signals and the site of polyadenylation (Fig. 6b). The polyadenylation signal, ATTAAA, found in pCSK9 and 12 is situated 13 and 14 bases, respec- tively, from the poly(A) tail and is a variation of the signal, AATAAA, originally observed by Proudfoot and Brownlee [27]. The ATTAAA sig- nal had also been reported for the chick lysozyme gene [28], mouse pancreatic a-amylase gene [29] and some human leukocyte interferon genes [30]. Since the polyadenylation signals from all the feather keratin genes sequenced thus far were of the type AATAAA [9], this may be indicative of some post-transcriptional control in the expression of feather and scale keratin genes such as mRNA turnover during development [31].

The clones pCSK9 and 12 are currently being used to probe a chicken genomic library in studies on the fine structural arrangement of the scale B-keratin genes.

Acknowledgements

The work reported here was supported in part by a grant under the Australian Research Grant Scheme. A Postgraduate Research Scholarship (to S.D.W.) was awarded by the Wool Research Trust Fund on the recommendation of the Australian Wool Corporation.

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