From the Divisions of Biochemistry of
Tissue-specific Regulation, ¶ Cell Biology, and
Resource
Center for Human Genome Research, German Cancer Research Center,
69120 Heidelberg, Germany
Received for publication, January 24, 2001
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ABSTRACT |
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Low stringency screening of a human P1 artificial
chromosome library using a human hair keratin-associated protein
(hKAP1.1A) gene probe resulted in the isolation of
six P1 artificial chromosome clones. End sequencing and
EMBO/GenBankTM data base analysis showed these clones to be
contained in four previously sequenced human bacterial artificial
chromosome clones present on chromosome 17q12-21 and arrayed into two
large contigs of 290 and 225 kilobase pairs (kb) in size. A fifth,
partially sequenced human bacterial artificial chromosome clone data
base sequence overlapped and closed both of these contigs. One end of
this 600-kb cluster harbored six gene loci for previously described human type I hair keratin genes. The other end of this cluster contained the human type I cytokeratin K20 and
K12 gene loci. The center of the cluster, starting 35 kb
downstream of the hHa3-I hair keratin gene, contained 37 genes for high/ultrahigh sulfur hair keratin-associated proteins
(KAPs), which could be divided into a total of 7 KAP multigene families
based on amino acid homology comparisons with previously identified
sheep, mouse, and rabbit KAPs. To date, 26 human KAP cDNA clones
have been isolated through screening of an arrayed human scalp cDNA
library by means of specific 3'-noncoding region polymerase chain
reaction probes derived from the identified KAP gene sequences. This
screening also yielded four additional cDNA sequences whose genes
were not present on this gene cluster but belonged to specific KAP gene
families present on this contig. Hair follicle in situ
hybridization data for single members of five different KAP multigene
families all showed localization of the respective mRNAs to the
upper cortex of the hair shaft.
The mature hair fiber is made up mainly of two major cell types.
An external sheath of overlapping flattened cuticle cells, often only a
single layer thick, encase the multicellular cortex. In distinct hair
fibers there is also a third cell type present in the centrally located
medulla. The proliferative cells that give rise to the mature hair
fiber reside in the bulb at the base of the follicle. As they leave the
germinative compartment, trichocytic differentiation begins, and in
matrix, cuticular, and cortical cells, the genes for two families of
structural proteins are activated (1). These families comprise the hair
keratins and the hair keratin-associated proteins. The human hair
keratin family consists of at least 16 members that are divided into an
acidic, type I, and a basic to neutral type II subfamily (2, 3). To
fulfill their biological function, specific type I and type II hair
keratin pairs are sequentially expressed and assembled into 10 nm
heteropolymeric keratin intermediate filaments, termed
KIFs.1 At the height of the
lower to middle cortex, these filaments are embedded into a matrix that
is formed by the hair keratin-associated proteins, termed KAPs. Based
on amino acid composition, essentially three classes of KAPs have been
described, the high sulfur KAPs (<30 mol % cysteine content), the
ultrahigh sulfur KAPs (>30 mol % cysteine content), and the high
tyrosine/glycine KAPs. Up to now, these three classes have been further
subdivided into 15 distinct KAP multigene families, based on amino acid
homologies and the nature of their repeat structures. Thus, the KAP1-3
and KAP10-15 families belong to the high sulfur KAPs (1, 4-14); the
KAP4,5,9 families encompass ultrahigh sulfur KAPs (15-20), and the
KAP6-8 families comprise tyrosine/glycine-rich KAPs (21-23).
The genes of individual KAPs of a distinct family were identified in
various species, and the localization of more than one KAP gene on a
given DNA fragment suggested the clustering of these genes in mammalian
genomes (4, 7-9, 12, 15, 17). Thus, in both sheep and man, a gene of
the KAP1 family has been identified on chromosome 11q25 and the
syntenic chromosome 17q12-21, respectively, regions also known to
contain sheep and human type I cytokeratin genes (4, 24, 25). Moreover,
human KAP5 genes could be assigned to chromosome 11p15 and
11q13 (26), and a cluster of high tyrosine/glycine KAP genes has been
identified on chromosome 21q22.1 (27).
The premise that KAP genes are clustered in the mammalian genome has
prompted us to screen a human P1 Artificial Chromosome Library (PAC
library) using a conserved probe of human KAP1.1A (4). The
PAC clones isolated lead to the identification of contiguous DNA
sequences in the EMBO/GenBankTM containing 37 genes for
high and ultrahigh sulfur KAPs, which could be classified into seven
KAP multigene families. cDNA transcripts of 30 KAP genes from these
families were isolated from an arrayed human scalp cDNA library.
In situ hybridization, using 3'-noncoding region probes from
single members of the human KAP1-3, -4, and -9 families showed
expression of the respective mRNAs in the differentiated portion of
the hair cortex.
Isolation of KAP Gene Containing PAC Clones--
A 764-bp
genomic PCR product of human KAP1.1A (hB2A) (4) (see
Table I) was used as a
hybridization probe to screen an arrayed human PAC library cloned into
the PAC vector AD10SacBII (library and screening by Genome Systems,
Inc., St. Louis, MO). Six clones, termed PAC7-PAC12, were isolated
(see Fig. 1). Initially, PAC DNA was prepared using a DNA midiprep kit
(Qiagen, Hilden, Germany), and the ends of the isolated PAC
clones were sequenced using a 33P chain termination cycle
sequencing kit as described previously (Amersham Pharmacia Biotech)
(3).
Arraying and Screening of a Human Scalp cDNA
Library--
Screening of a human scalp cDNA library cloned into
the
The arrayed human scalp cDNA library filters were screened using
150-250-bp 3'-noncoding region probes from the putative hair keratin-associated protein genes discovered in the
EMBO/GenBankTM data base BAC clones (see Table I).
Prehybridization/hybridization was performed using 5× SSC, 5×
Denhardt's solution, 0.1% sodium pyrophosphate, 1% SDS as
hybridization buffer at 59 °C overnight. Post-hybridization washes
were performed 3 times using 0.5× SSC, 1% SDS at 59 °C for 30 min.
The filters were autoradiographed using Kodak XAR5 film (Amersham
Pharmacia Biotech). The position of doubly positive clones on
the filter was determined using a 5 × 5 scoring pattern, and the
coordinates from the scoring pattern were then used to obtain the
correct clone from the scalp library (the clones described in Table
II are available from the German Resource
Center or from the authors).
Subcloning and Characterization of PAC10--
The
subcloning of PAC NdeI restriction enzyme fragments has been
described previously (2). The subcloning of PAC10 resulted in the
isolation of ~80% of the total NdeI fragments. End
sequencing of the isolated NdeI subclones with subsequent
EMBO/GenBankTM analysis of these DNA sequences identified
many of these sequences on the draft DNA sequence (in progress
sequencing) of two BAC clones (accession numbers AC025904 and
AC037482). At the time of analysis these clones were 174,032 and
180,944 bp in size and consisted of 17 and 30 fragments, respectively.
Comparison of these draft sequences with the subcloned PAC10 sequences
allowed a complete orientation of the relevant draft sequences on PAC10.
Automated Fluorescent DNA Sequencing--
Sequencing of the
clones isolated from the arrayed human scalp cDNA library as well
as the NdeI subclones from PAC10 were performed by
fluorescence dye terminator cycle sequencing using the Big Dye DNA
Sequencing Kit (Applied Biosystems, Inc., Weiterstadt, Germany)
according to the manufacturer's instructions (see also Ref. 3). The
sequencing reactions were analyzed on a ABI310 capillary DNA sequencing
apparatus using the 47-cm capillary.
DNA and Protein Analysis--
DNA analysis of PAC, BAC, and
cDNA clones was done using the Wisconsin GCG software package
(version 10) as contained in the Heidelberg Unix Sequence Analysis
Resource. The initial KAP EMBO/GenBankTM data base searches
were performed using the BLASTN program. KAP homology searches on
individual BAC clones was accomplished using the SIMILARITY program.
Multiple KAP protein homology comparison were done using the CLUSTAL
program. Repeat structure analysis was performed using the program
DOTPLOT. cDNA sequence assembly and correction was accomplished
using the STADEN program package.
In Situ Hybridization--
In situ hybridization
(ISH) of cryostat sections of human hair follicles containing scalp
(taken for medical reasons) were performed as described previously
(29-31). 35S-Labeled cRNA transcripts of hKAP1.5, hKAP2.4,
hKAP3.3, hKAP4.3, and hKAP9.2 derived from subcloned 3'-noncoding
region PCR probes (see Table I) were used for detection of the
respective KAP mRNA species. Specific ISH signals were visualized
using a confocal laser scanning microscope (LSM 510; Carl Zeiss, Jena,
Germany). Simultaneous visualization of reflected ISH signals through
epi-illumination and transmitted light in bright field were combined by
overlay using pseudocolors in Figure 8 (transmission image
green, electronically changed to black/white; reflection image (ISH
signal) shown in red).
Discovery of a Domain of High/Ultrahigh Sulfur KAP
Genes--
Screening of an arrayed human PAC library with a
full-length probe of the human KAP1.1A gene (4) leads to the
isolation of six PAC clones, termed PAC7-12. DNA end sequences derived
from each of these clones were compared with sequences in the
EMBO/GenBankTM data base, which resulted in the
identification of four fully sequenced BAC clones, AC003958, AC006070,
AC007455, and AC00423, representing two DNA contigs on chromosome
17q12-21. Contig 1, comprising BAC clones AC00395 and AC006070, covered ~290 kb, and contig 2, consisting of BAC clones AC007455 and AC00423,
was 223 kb in size (Fig. 1). In addition,
two partially sequenced BAC clones, AC025904 and AC037482 (not shown in Fig. 1), were also identified. Clone AC025904 was, at the time of
writing, ~170 kb in size and consisted of 17 fragments. Six of these
fragments exhibited sequence identity with AC006070 and covered about
80 kb of one end of contig 1, whereas another fragment corresponded to
~2.2 kb of one end of contig 2, i.e. BAC clone AC007455.
Thus, BAC clone AC025904 connected contigs 1 and 2 to each other (Fig.
1). The remaining 10 AC025904 fragments, comprising ~101 kb of DNA,
which partially covered the gap between contigs 1 and 2 (Fig. 1),
showed low sequence identity to both contigs. The second, partially
sequenced BAC clone, AC037482, ~180 kb in size, which overlapped with
~23.7 kb of one end of AC000423, progressed completely through
AC007455 and ended in the gap between contigs 1 and 2, was also used
for fragment identification and orientation in the region between the
two contigs.
Identification and Characterization of KAP Genes--
Homology
analysis of all the genomic DNA sequences described above, using the
human KAP1.1A gene open reading frame (ORF) sequence (4) for
comparison, led to the identification of 37 putative KAP gene loci. The
corresponding KAP gene cluster covered a region of ~300 kb (Fig. 1).
As a rule, the KAP genes were ~1 kb in size, consisted of one single
exon, and were separated by 2.5-38 kb of intervening sequences.
Furthermore, no unified direction of transcription could be found. Four
of the loci,
Screening of an arrayed human scalp cDNA library using 3'-noncoding
region probes derived from the 37 individual KAP genes led to the
isolation of 30 novel KAP cDNAs (Table II). Only 26 corresponded to
KAP gene sequences present in the cluster shown in Fig. 1. Thus the
combination of human genomic/cDNA sequences analyzed in this report
and by others (4) makes a total of 43 human KAP sequences characterized
at the gene or cDNA level.
Amino acid translation of all the KAP genomic or cDNA sequences and
comparison with the protein sequences of known KAPs from sheep, mouse,
rabbit, and human via amino acid multialignment and evolutionary tree
formation (Clustal and Clustree) allowed the assignment of the human
KAPs to either the high sulfur KAP families KAP1, -2, -3 or the
ultrahigh sulfur KAP families KAP4 and -9. These five multigene
families were grouped together in a contiguous gene region shown in
Fig. 1. Two single KAPs, which did not possess high homology with any
other known KAP family member, were termed hKAP16.1 and hKAP17.1 (Fig.
1).
The human high sulfur KAP1 gene family found on this DNA contig
composed of four genes, hKAP1.1B, hKAP1.3,
hKAP1.4, and hKAP1.5 (Figs. 1 and
2). Two previously isolated human KAP
genes, located on a single genomic clone and termed hB2A and
hB2B (4), were renamed hKAP1.1A and
hKAP1.2 in this paper to reflect changes that have recently
occurred in KAP nomenclature (1). Neither of these hKAP1 genes has been
found on the DNA domain of Fig. 1 nor could an hKAP1.2 cDNA be
isolated. The hKAP1.1A and hKAP.1B genes are
nearly identical in DNA sequence. The hKAP1 proteins are 12.3-18.2 kDa
in size and contain 24.1-25.7 mol % cysteine (Table II). The members
of the hKAP1 family exhibit two unique, highly conserved motifs at the
beginning and in the center of each coding region (Fig. 2). In
addition, a 20-amino acid subdomain downstream of this region
(ASCCRPSYCGQSCCRP(A/V)CCC) is completely conserved in all species
examined. The KAP1 proteins contain a variable number of pentapeptide
repeats (CCQ(P/T)S, CCETS), and a specific combination thereof (CCETS
CCQPS) shows similarity to the decapeptide repeat (SIQTSCCQPT)
described in the sheep KAP1 family (1, 7, 8).
All KAP family members analyzed appear to have unique, family-specific
amino- and carboxyl-terminal regions (see below). For the hKAP1 family,
these sequences are M(A/T)CCQT (amino terminus) and (C/S)EPTC (carboxyl
terminus). Two exceptions are hKAP1.4, which does not possess the
amino-terminal homology, and hKAP1.2, which lacks the carboxyl-terminal homology.
Five human high sulfur KAP2 genes, hKAP2.1A, hKAP2.1B,
hKAP2.2, hKAP2.3, and hKAP2.4, and one hKAP2
pseudogene,
hKAP2 proteins are 13.48-13.51 kDa in size, and their cysteine content
(27.3-28.1 mol %) is slightly higher than that of the hKAP1 proteins
(Table II). They exhibit a high amino acid sequence homology to each
other, as well as to two known sheep KAP2 sequences (6, 14) (Fig.
3). hKAP2 proteins consist of a series of
repetitive pentamers in their amino- and carboxyl-terminal domains,
which are separated by a KAP2-specific central amino acid region (Fig. 3). The pentamer repeats consistently start with double cysteines. Similar to the hKAP1 family, the amino and carboxyl termini of hKAP2
members appear to be family-specific sequences (amino terminus, MTGSCC;
carboxyl terminus, CRTSSC).
The human high sulfur hKAP3 gene cluster contained four members,
hKAP3.1, hKAP3.2, and hKAP3.3, and
pseudogene
Although at present only two ultrahigh sulfur KAP4 family members
are known from sheep and rabbit (15, 16), the human KAP4
gene family turned out to be the largest KAP family located on the DNA
domain shown in Fig. 1. It is composed of 15 genes, hKAP4.1-hKAP4.15, as well as one pseudogene,
The ultrahigh sulfur hKAP9 family turned out to be the second largest
family found. It consisted of seven gene sequences, hKAP9.1-9.7, two cDNA sequences not found on the DNA
contig (hKAP9.8-hKAP9.9), and one pseudogene
Based on their cysteine content, the proteins encoded by the two novel
KAP genes hKAP16.1 and hKAP17.1 (Figs. 1 and
7) were identified as high sulfur
(hKAP16.1) or ultrahigh sulfur KAPs (hKAP17.1), respectively (Table
II). Remarkably, they constitute both the largest (hKAP16.1, 53.9 kDa)
and the smallest (hKAP17.1, 9.5 kDa) of all hKAPs whose genes are
located on the cluster described in Fig. 1. Screening of the cDNA
library yielded only a cDNA clone for hKAP17.1.
Hair Follicle KAP Expression--
Radioactive in
situ hybridizations using 35S-labeled 3'-noncoding
region probes derived from representative members of each multigene KAP
family found on the DNA contig (hKAP1.5, hKAP2.4, hKAP3.3, hKAP4.3, and hKAP9.2) were
performed on cryosections of human scalp epidermis (a detailed
expression study of all KAP members is in
progress).2 Four of the
probes were specific for each of the respective genes analyzed
(hKAP1.5, hKAP2.4, hKAP3.3, and
hKAP4.3). A unique probe for the hKAP9.2 gene
could not be found. The probe used also showed very high sequence
identity to the hKAP9.4 and hKAP9.5 genes. The
expression pattern for hKAP9.2 shown in Fig.
8e probably represents the
expression patterns of all three genes. All five KAP family members
tested showed their respective mRNA expression specifically in the
middle/upper portions of the hair cortex, in the region termed the
keratogenous zone (Fig. 8). No hybridization signals could be found in
the hair matrix and cuticle, as well as the inner or outer root sheath
for any of the KAP family members assayed. In addition, hKAP
1.5 and hKAP9.2 showed no obvious expression in the
medulla of the hair follicle. Medullar expression could not be
determined for hKAP1.5, hKAP2., and
hKAP3.3 because no clearly medullated hair were found in
these samples. In general, the ISH signal intensity was relatively
uniform among all of the KAP family members.
In the present study we have characterized an ~300-kb DNA
contig, which harbors 37 members of the human hair KAP multigene family. Moreover, we were able to show that this gene cluster is
embedded in the type I keratin gene domain on chromosome 17q12-21 (Fig.
1). Adjacent to one end of the KAP gene cluster lie six genes
(hHa7, In the region between these two keratin gene domains, on BAC
clones AC006070, AC025904, AC037482, and AC007455 and partially on
clone AC003958, we identified a cluster of 37 KAP genes. They can be
grouped into three high sulfur KAP families, hKAP1, hKAP2, and hKAP3,
two ultrahigh sulfur families, hKAP4 and hKAP9, and two single
high/ultrahigh sulfur hKAP genes, hK16.1 and
hKAP17.1. Due to the partial nature of BAC clone AC025904, which connects the other two fully sequenced genomic contigs, neither
the exact size of this KAP gene cluster nor the exact number of KAP
genes in this cluster is known. An approximation of the completeness of
clone AC025904, can be made, however. The size of AC025904 has been
estimated at 170 kb by pulse field gel electrophoresis (see EMBO
accession file), and the amount of DNA sequenced thus far is 172,432 bp, which would suggest that this BAC clone sequence is probably over
90% complete. In addition, the identification of four further KAP
cDNAs, hKAP4.13, hKAP4.15, hKAP9.8 and hKAP9.9, whose genes do not
lie on the current genomic sequence, but structurally belong to their
respective families, increases the probability that the majority of the
KAP genes in the 300-kb region have been elucidated. On the other hand,
this finding suggests that further individual KAP genes or gene
clusters of some of the families described above should be present in
other chromosomal locations. This also includes the previously
described hKAP1.1A and hKAP1.2 genes (4), which
have not been found on the current contig. An EMBO data base search
using hKAP1.2, hKAP4.13, hKAP4.15, hKAP9.8, and
hKAP9.9 genomic/cDNA sequences did not, however, reveal
further genomic clones that could harbor these sequences.
The high/ultrahigh sulfur hKAP genes were essentially identified by
their homology with previously described KAP gene sequences from other
species. In order to affirm that the gene loci identified were
transcribed members of the hair KAP family, an attempt was made to
isolate their respective cDNA sequences from an arrayed human scalp
cDNA library. cDNA library screening was chosen over direct
amplification of hair follicle mRNA by reverse transcriptase-PCR, considering that the lack of intron sequences in the KAP genes would
not allow discrimination between true reverse transcriptase-PCR amplification products and the artifactual amplification of genomic DNA
sequences. The isolation of cDNAs by this method, however, was
partially limited by the conservation of sequences among KAP family
members in their 3'-noncoding regions. For example hKAP9.2 and hKAP9.4-hKAP9.9 were all fairly homologous to each
other in these regions. Thus, screening with one member of a family
often leads to the isolation of another member that exhibited stronger expression patterns. This conservation of sequence also lead to the
fortuitous isolation of related cDNAs not, as yet, found on the
genomic sequences analyzed (i.e. the cDNAs for
hKAP4.13, hKAP4.15, hKAP9.8, and hKAP9.9). Weak expression patterns
would also explain why several KAP family members (hKAP1.4,
hKAP2.3, hKAP9.1, hKAP9.6, hKAP9.7, and hKAP16.1) were not found, for
the arrayed scalp library used in these studies consisted of only
26,000 clones and therefore allowed only detection of moderate to high
KAP cDNA expression.
Protein homology comparisons of the translated hKAP gene/cDNA ORFs,
with previously described KAP families (1), allowed a clear-cut
division of most of the hKAP proteins into five distinct families.
Historically, the initial division of high/ultrahigh sulfur KAPs into
families and their numerical assignments according to the proposed KAP
nomenclature were based on their mol % cysteine content, their
homology to each other, and to a certain degree, the nature of their
repeat structures (1). However, recent and simultaneous publications of
various murine KAPs has lead to confusion in the current KAP
nomenclature.3 In 1998, Takaishi et al. (11) isolated a mouse KAP expressed in the
periderm of embryonic day 16.5 mouse skin but not found in the mouse
hair follicle. Its cysteine-rich structure, however, as well as its
presence in the filiform papillae of the tongue and in tail scale
epidermis led to the conclusion that it is a member of the high sulfur
KAP family, termed mKAP13.1 (11). At the same time, Aoki et
al. (13) described a high sulfur KAP cDNA found in mouse hair
follicles. The authors also named this protein mKAP13, but currently,
it is designated in the data base as mKAP14.1. Thereafter, two
additional mouse KAP genes were identified, termed pmg1 and
pmg2 (12). Pmg1 exhibits 94% amino acid identity with the
protein described by Aoki et al. (13) and thus should be
named mKAP14.2. Pmg2, in contrast, exhibits only 43% identity with
pmg1 (i.e. mKAP14.2) (12) or the current mKAP14.1
(13), and these are the KAPs with the highest homology to pmg2.
Therefore, pmg2 should be designated mKAP15.1. As a consequence, the
two single hKAP genes identified in this study, which did not readily fit into the schemes of the known KAP gene families, were named hKAP16.1 and hKAP17.1.
hKAP16.1 is the largest high sulfur KAP found to date (517 amino
acids). In general, hKAP16.1 possesses a fairly low homology to the
other KAP family members (~35-48% identity). The highest identity
is seen with the sheep high sulfur KAP10 (48.2%), also one of the
largest KAP proteins known (294 amino acids) (1).
Multiple alignments among any member of one KAP family give a much
higher degree of homology (~60-98%) than comparisons between sKAP10
or hKAP16.1 and these families. As such, hKAP16.1 should be considered
as a member of a separate high sulfur KAP family. The ultrahigh sulfur
hKAP17.1 is a very small protein (9.1 kDa) and contains a central
cluster of cysteine/glycine repeats, very similar to members of the
ultrahigh sulfur KAP5 family (1, 18, 20). The amino acid homology
between hKAP17.1 and the known KAP5 family members is, at most, 47.8%,
whereas the range of sequence identity between the known KAP5 members
is in the order of 56-76%. In addition, all members of the KAP5
family isolated so far contain a highly conserved 14-mer amino acid
sequence (P/TCCC/-VPACSCCSSC) not present in hKAP17.1.
It should be emphasized that the division of the human KAPs into
distinct families was facilitated by bioinformatic means. Multiple
amino acid alignments of all the KAPs isolated here were used in
combination with the previously described KAP proteins from several
species in order to generate a single evolutionary tree of all known
KAP proteins (Fig. 9). This procedure
allowed a graphic representation of homologous family members that
neatly grouped previously characterized family members together with the human members found on the genomic contig characterized in this
paper. Further Clustal alignment of the known/new family members
affirmed the validity of this approach (see Fig. 2-6). Additionally, this tree grouped together several additional KAP family members from
other species, thus indicating families that might, perhaps, also be
clustered together on the mammalian genome. Although this procedure is
statistically insufficient for evolutionary tree analysis due to KAP
protein size variability, it appears in this case to be a reliable tool
for the division of KAPs into families.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Primers used for the generation of hybridization probes for KAP
cDNA screening
-ZapII vector was accomplished as described previously (28). At a later date, arraying of single bacterial phagemid clones into microtiter plates and the preparation of DNA hybridization filters from
these single clones were performed. Briefly, a mass in vivo excision/conversion of the human scalp library was accomplished, converting the
-library clones into a more manageable Bluescript phagemid form. Dilutions of this excised library (~6000 clones) were plated onto 22 × 22-cm LB agar plates under
ampicillin selection, and single colonies were picked and arrayed into
132-well microtiter dishes using a spotting robot. Approximately 26,000 of the single clones were then double-spotted onto a single, gridded 22 × 22-cm nylon membrane in an ordered fashion. The single
colonies were grown on these filters overnight and lysed, and their DNA was cross-linked to the nylon membrane (for further details see the
German Resource Center Web site).
GenBankTM accession numbers for genomic and cDNA sequences
presented in this study
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Physical map of the human KAP gene
cluster with its flanking hair keratin/cytokeratin genes.
Black horizontal lines represent sequenced BAC clones found
in the EMBO/GenBankTM data base. The dotted line
represents an area covered by the partially sequenced BAC clone
AC025904 that closes the gap between the completely sequenced BAC
clones. Orange horizontal lines show PAC clones isolated in
this laboratory. Red boxes indicate the position of
individual KAP genes; green boxes show the position of
keratin gene/pseudogene/orphan exon loci. Horizontal black
arrowheads indicate the direction of gene transcription. The
long double-headed arrows delineate the two finished contigs
referred to in the text. The blue bar indicates a region
closed by long range PCR. The numbers below the red
boxes refer to the names of the respective KAP genes (for example
9.5 = hKRTAP9.5). Numbers in bold
represent gene loci for which a respective cDNA has been found. The
text below the horizontal black solid/dotted lines indicates
the BAC clone accession numbers and the size of each clone in bp. The
text above the fragments covering BAC clone AC025904 (for
example x14, a29) represents relevant individual DNA contigs
on this clone (shown by x plus a number, i.e.
x14) as well as on the partially sequenced BAC clone
AC037482 (a plus a number, i.e. a29).
The accession numbers for the hair/cytokeratin gene/pseudogene
sequences are hHa7 (Y16793), hHaA
(Y16795), hHa1 (Y16787), hHa4 (Y16790),
hHa3-II (Y16789), hHa3-I (Y16788), K20
(X73501), and K12 (D78367). The accession number of the
putative transcribed keratin pseudogene cDNA is AL117538.
KAP2A,
KAP3A,
KAP4A, and
KAP9A, showed pseudogene character based on
the presence of premature stop codons or frameshifts in the coding
regions of the genes.
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Fig. 2.
Multiple sequence alignment of hKAP1 family
members. All sequences shown are open reading frame translations
of the gene sequences (see Table II). The multialignment was performed
using the Clustal program (39). The asterisks
beside the protein names indicates KAP1 members from other
species (s, sheep; r, rat). Asterisks
below the alignment indicate sequence identity; dots
denote sequence homology. Hyphens in the amino acid
sequences show gaps introduced during alignment. The original names of
the previously isolated hKAP1.1A and hKAP1.2
genes are shown in brackets. The names of human KAP family
members in bold type refer to gene products for which a
respective cDNA sequence has been found. Solid boxes
frame the putative pentapeptide repeats described in the text. The
dotted boxes show the decapeptide repeats described in sheep
KAP1 family proteins (1, 7, 8). The hatched boxes
below the sequence indicate conserved, non-repetitive
regions. Accession numbers for sheep and rat KAP sequences are as
follows: sB2B, kr2b_sheep.sw; sB2C,
kr2c_sheep.sw; sB2D, kr2d_sheep.sw; rB2E and
rB2F, AB003753.
hKAP2A, were identified (Fig. 1).
The respective cDNA sequences to four of the genes
(hKAP2.1A, hKAP2.1B, hKAP2.2, and
hKAP2.4) could be isolated (Table II).
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Fig. 3.
Multiple sequence alignment of
hKAP2 family members. The identical amino acid sequences of hKAP2A
and hKAP2B are shown only once. For details, see legend to Fig. 2.
Accession numbers for sheep KAP sequences are as follows:
sBIIIA3, kra3_sheep.sw; sBIIIA3A,
kr3a_sheep.sw.
hKAP3A (Fig. 1). The cDNAs of
the three functional genes could be identified. The KAP3 proteins are
10.3-10.5 kDa in size and have a rather low cysteine content of
18.3-19.4 mol % (Table II). Like the KAP2 family members, the human
KAP3 family members also display high homology among each other and
possess an identical number of amino acids (Fig.
4). hKAP3 family members as well as their
known orthologs of other species (1, 6, 9) do not exhibit a discernible repeat structure and exhibit a weaker head and tail sequence
specificity (Fig. 4).
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Fig. 4.
Multiple sequence alignment of hKAP3 family
members. For details see legend to Fig. 2. Accession numbers for
sheep KAP sequences are as follows: sBIIIB2, m21099;
sBIIIB3, krb3_sheep.sw; sBIIIB4,
krb4_sheep.sw.
hKAP4A. Thirteen members have been identified
as cDNA sequences (Table II). However, the genes corresponding to
the cDNA hKAP4.13 and hKAP4.15 could not, as yet, be found on the
isolated DNA domain (Fig. 1). The size of the encoded proteins ranges
from 11.5 to 29.7 kDa, and their cysteine content varies between 33.6 and 36.8 mol % (Table II). The hKAP4 members exhibit a highly
conserved amino-terminal end region, MV(S/N)SCC, followed by a central
region of highly repetitive, di-cysteine-containing pentamers,
separated by occasional pentameric, non-repetitive segments (Fig.
5). In contrast to the hKAP1-3 families,
no large, non-repetitive regions are present in the central domain of
hKAP4 members. The carboxyl-terminal end domains of hKAP4 proteins are
less conserved, but many members contain a terminal CC(G/A)SSCC
sequence (Fig. 5).
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Fig. 5.
Multiple sequence alignment of hKAP4 family
members. KAP proteins that possess a high amino acid identity are
shown in a common color. +hKAP4.13 and hKAP4.15 are amino acid
sequences derived from cDNA clones. For details, see legend to Fig.
2. Accession numbers for sheep and rabbit KAP sequences are as follows:
sKAP4.1, X73462; rbKAP4.2, X80035.
hKAP9A (Figs. 1 and
6). The size of the hKAP9 members ranges
from 16.3 to 26.3 kDa, and their cysteine content varies from 31.3 to
35.6 mol % (Table II). The structure of the hKAP9 family members is,
in principle, reminiscent of that of the KAP2 family in that amino- and
carboxyl-terminal pentad repeat structures are separated by central,
conserved and hKAP9-specific amino acid sequences. Similar to the KAP4
family, the repetitive elements consist of multiple double
cysteine-containing repeats, interrupted by two larger nonrepetitive
regions of 14 and 33 amino acids. Like the other KAP families, KAP9
family members display conserved amino- and carboxyl-terminal amino
acid sequences (amino terminus, MT(H/N); carboxyl terminus,
CCQ(PHS)(SAF)SCC.
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Fig. 6.
Multiple sequence alignment of hKAP9 family
members. For details, see legend to Fig. 2. +, hKAP9.8 and hKAP9.9
are amino acid translations of full-length cDNA sequences.
Accession numbers for mouse KAP sequences, mKAP9.1,
M27685.
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Fig. 7.
Amino acid sequence of hKAP16.1
(A) and hKAP17.1 (B). An
asterisk at the end of the amino acid sequence indicates a
stop codon.
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Fig. 8.
ISH studies of KAP family members.
Longitudinal section of human scalp hairs were hybridized with
3'-noncoding region probes as follows: a, hKAP1.5;
b, hKAP2.4; c, hKAP3.3; d, hKAP4.3;
and e, hKAP9.2. Inserts in c and
e show representative cross-sections of the specifically
expressed KAP mRNA. Red arrows mark the main region of
mRNA expression. dp, dermal papilla; ors,
outer root sheath; irs, inner root sheath; co,
cortex; cu, hair cuticle; med, medulla.
Bars in panels are 150 µm.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
hHaA, hHa1, hHa4, hHa3-II, and
hHa3-I) that are part of the previously described type I
hair keratin gene domain (3). On the other side of the KAP gene
cluster, we identified the genes for cytokeratins hK20 and hK12 (32,
33). These functional genes are, however, preceded by an orphan exon
and a transcribed pseudogene that corresponds to the EMBO data base
cDNA sequence AL117538, originating from a human testis cDNA
library (see accession file). This gene contains 7 exons and six
introns, with the last six exons displaying a high homology to the
corresponding exons of the adjacent hK12 and hK20
genes, whereas exon one differs completely from keratin gene sequences.
The gene possesses an open reading frame for a truncated keratin,
featuring the loss of the highly conserved helix initiation motif that
is indispensable for correct intermediate filament formation (34). This
suggests that the gene represents a transcribed type I keratin
pseudogene. The gene is thus reminiscent of a similarly truncated form
of the human type II hair keratin gene, hHb1, which contains
exons 5-8 of this gene and which is expressed aberrantly in breast
tumors (35). The truncated hHb1 protein has been shown to be
synthesized in these tumors (36).
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Fig. 9.
The division of keratin-associated proteins
into families via Clustree. A multiple alignment of the complete
sequences of all known high/ultrahigh sulfur KAP proteins as well as
KAP proteins found in this report was performed using the program
Clustal (39). The segregation of the previously known multialigned
family members with the KAP proteins described in this paper was
performed using the program Clustree (40). KAPs named in
black are the proteins described above that segregated with
known KAP family members from other species. KAPs named in
purple, hKAP16.1 and hKAP17.1, denote two proteins that did
not segregate well with known family members. KAPs named in
red represent proteins found in other species. KAPs named in
green denote previously described human KAP proteins. This
multiple tree alignment via Clustal was adequate enough for division of
the KAP proteins into families. It was not statistically significant
enough to determine paralogous evolutionary relationships. Additional
accession numbers not listed in Table II or Figs. 2-6 are as
follows: sB2A, m21099; mSER-UHSP1, m37759;
mSER-UHSP2, m37760; hKERA, AJ006692;
hKERB, AJ006693; hKAP5.1/UHSP3, x55293;
sKAP5.1 and sKAP5.2, x55294; sKAP5.4,
x73434; sKAP5.5, x73435; mKAP11.1
(hacl1), u03686; mKAP12.1, af081797;
mKAP13.1 (mEMB-UHSP1), af031485;
mKAP14.1, d85925; mKAP14.2
(pmg1) and mKAP15.1 (pmg2),
af162800. The amino acid sequence for sKAP10.1 can be found in
Powell and Rogers (1).
The presence of cysteine-rich repeat structures has been described in most of the previously characterized KAP proteins. These structures often contain double or triple cysteines, and several of these proteins (mostly of the KAP4 family) possess serine or threonine doublets (1). Decapeptide repeat structures have been shown in several KAP1 family members from other species (1, 7, 8) as well as in the single member of the mKAP9 family (1). Pentapeptide repeats have been shown in amino acid fragments from members of the sheep KAP1 and KAP2 families (6), and frequently occurring pentameric repeats of the form CC(R/Q)P(S/T) have been found in sheep and rabbit KAP4 family members (1, 15, 16). In contrast, little repetitiveness of structures is seen in the KAP3 family (1, 9). In humans, repeat structures of the KAP1, -2, -4, and -9 family members consist essentially of amino acid variations of a double cysteine-containing pentapeptide. This pentapeptide structure is strikingly visible upon analysis of KAP sequences using the program DOTPLOT, which graphically illustrates this repetitiveness. The degree of pentapeptide repetitiveness in the human KAP1 family ranges from 9 to 16 elements. Two types of these elements, CCQPS and CCETS, correspond, to a certain degree, to the 10-member repeat elements previously reported in sheep and rat (1, 7, 8). In humans, however, this element is not as highly conserved. The double cysteine pentapeptide members of the human KAP2 family show 10-11 repeats and appear to have more variation in the third or the fifth amino acid when compared with sheep KAP2 members (6). The degree and absoluteness of structure in the central portions of all KAP4 family members are striking. This subdomain consists of nearly invariant double cysteine-containing pentameric repeats. Many of these repeats contain serine/threonine or proline in position 4 and primarily serine and threonine in position 5. The degree of repetitiveness in the hKAP4 family is high (24-29 elements). The hKAP9 family members also contain pentameric repeat structures (14-18) that largely resemble the structures found in the hKAP4 family. Similar to sheep KAP3 (6, 9), the human KAP3 orthologs contain minimal repeat structures.
In addition to repeat structures, members of the hKAP1, hKAP2, and hKAP9 families contain central, highly conserved non-repetitive structures that are specific for each family. In the hKAP1 family, these are two regions of 21 and 43 amino acids (see hatched boxes in Fig. 2). These regions are also largely conserved in sheep and rat. In the hKAP2 family, this unique, non-repetitive structure is 24 amino acids long and is highly conserved (see hatched box, Fig. 3). The hKAP9 family non-repetitive regions are 14 and 33 amino acids in length and, with the exception of hKAP9.1 which does not appear to contain a non-repetitive region, are also highly conserved (see hatched boxes, Fig. 6).
The degree of DNA/amino acid sequence conservation between members of one hKAP family is often quite strong. This is especially the case among the hKAP2, hKAP3, and several of the hKAP4 family members. For example hKAP2.1A and hKAP2.1B, are completely identical in their open reading frame and thus are two genes encoding the same protein. These genes are, however, fairly divergent in their 5'- and 3'-noncoding regions. In addition, the open reading frame of hKAP2.2 varies in only one nucleotide when compared with hKAP2.1A/B, which results in the substitution of a cysteine for an arginine. The open reading frame and 3'-noncoding region of KRT2.2 and KRT2.1B are also nearly identical with the exception of a 39-bp insertion in the hKAP2.1B gene in a region located just downstream of the stop codon. The cDNA sequences for all three of these KAP2 genes have been found (Table II). A very similar case is seen in three members of the KAP4 family, hKAP4.8, hKAP4.11, and hKAP4.14. All three have a very high sequence identity both in their coding and non-coding regions. hKAP4.8 and hKAP4.14 possess, however, a 106-bp insert in their ORF which is not found in hKAP4.11. When compared with each other, the hKAP4.8 and hKAP4.14 proteins have the same number of amino acids as well as a high sequence identity at the DNA (95%) and protein (92%) level. cDNA sequences have been found for hKAP4.8 and hKAP4.14. Other members of the KAP4 family that show high amino acid homology among each other are shown as sequences of identical color in Fig. 5.
The high homology among members of a single KAP family coupled with the strong similarities in the cysteine repeat structure inside a family lead to the conclusion that multiple family members might have arisen by gene duplication, especially in duplications/deletions of the cysteine repeat motifs of these genes. The inability to delineate specific orthologous members between family members of the various species isolated to date might lead to the hypothesis that multiple members of a distinct family in a given species might have evolved independently from members in other species. These new members, however, probably originated from a common family-specific ancestral gene found in all mammals.
The presence of a high cysteine content in the high/ultrahigh sulfur KAPs has led to the assumption that these residues might play an important role in the bundling of KIFs via cysteine cross-links (1). Since the pentameric repeats of the hKAP1, -2, -4, and -9 families contribute to the majority of cysteines in these members, they probably are significant in this KIF bundling. This hypothesis is also consistent with the expression profile of each member of the hKAP1-3, -4, and -9 families analyzed. They show a unique and specific mRNA expression in the differentiated, highly keratinized portions of the hair cortex (Fig. 8). The expression pattern of the KAP1, -2, -4, and -9 family members largely reflects that seen in other species. For example, the cortical expression pattern of hKAP1.5 (Fig. 8, a and a') is similar to that seen by rat B2F (KAP1 family) (7). In rat, the absence of B2F signal in the medulla could be determined. For the KAP2 family, hKAP2.4 expression (Fig. 8b) is similar to that seen in one sheep KAP2 family member (1). Like hKAP4.3 (Fig. 8d), upper cortical expression of KAP4 family members has also been shown in rabbit (15) and sheep (16). All of the previously described species show KAP4 family member expression in the differentiated portions of the hair cortex. However, the expression in sheep, unlike rabbit and human, is localized exclusively to the paracortical cells of this region. In human hairs, a discrimination between ortho- and paracortex ("tight" versus "loose" packing of KIF bundles) is barely distinguishable. Transmission electron microscopy studies on Caucasian hair follicles largely show a central core of paracortical cells surrounded by orthocortical cells (37). Despite this pattern, no obvious ortho-/paracortical division of signal intensity could be seen in hKAP4.3 expression. hKAP9.2 expression (Fig. 8e) is similar to that seen in mouse mKAP9.1 (38). However, mKAP9.1 also showed a cuticular expression not seen for hKAP9.2. Finally, the upper cortical expression of hKAP3.3 (Fig. 8c) shows, for the first time, expression by a KAP3 family member.
The characterization of this domain of KAP family genes provides an
initial study for the complete characterization of the human
high/ultrahigh sulfur keratin-associated proteins. Several questions
remain unanswered concerning the completeness of the KAP multigene
families presented here, especially in respect to other chromosomal
loci that might harbor further KAP gene members of the families
described in this study. In addition, comprehensive mRNA and
protein expression studies of all KAP family members presented here are
needed in order to gain a better picture of how hair keratin-associated
proteins are expressed in the hair follicle. This should eventually
lead to functional studies of KAPs giving further insight into their
precise role in hair fiber formation.
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ACKNOWLEDGEMENTS |
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We thank Dr. Herbert Spring for help with confocal laser microscopy and Dr. Matthias Schick for help in the arraying of the human scalp cDNA library. We also thank the members of the German Resource Center/Primary Database, The German Resource Center for the Human Genome, Heidelberg and Berlin, Germany, for their help in the growing of the cDNA clones. The clones listed in Table II and human scalp cDNA library (library number 636) are available from the German Resource Center/Primary Database.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Biochemistry of Tissue-specific Regulation (B0501), German Cancer Research Center, 5th Floor, Rm. 522, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany. Tel.: 49-6221-423248; Fax: 49-6221-424406; E-mail: m.rogers@dkfz-heidelberg.de.
Published, JBC Papers in Press, February 27, 2001, DOI 10.1074/jbc.M100657200
2 L. Langbein, M. A. Rogers, S. Praetzel, H. Winter, C. Ehmann, and J. Schweizer, manuscript in preparation.
3 The evaluation presented here interprets the current data in Locus Links, the database of the National Center for Bio/Technology Information.
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ABBREVIATIONS |
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The abbreviations used are: KIF, keratin intermediate filament; KAP, keratin-associated protein; PAC, P1 artificial chromosome; BAC, bacterial artificial chromosome; ORF, open reading frame; PCR, polymerase chain reaction; kb, kilobase pairs; bp, base pairs; ISH, in situ hybridization; DSP, did not screen positive; NF, not found.
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