1
Institute of Genetics, Division of Molecular Genetics and Bonner Forum
Biomedizin, University of Bonn, 53117 Bonn, Germany
2
Max-Planck-Institute for Biophysical Chemistry, Department of Biochemistry,
37070 Goettingen, Germany
*
Authors for correspondence (e-mail:
t.magin{at}uni-bonn.de
;
m.hesse{at}uni-bonn.de
;
r.longo{at}gwdg.de
)
Accepted May 23, 2001
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SUMMARY |
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Key words: Human genome, intermediate filament proteins, keratins, lamins, neurofilament proteins, pseudogenes, disease
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INTRODUCTION |
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Gene mapping studies revealed that genes coding for non-keratin IF proteins
are not clustered (International Human Genome Sequencing Consortium,
2001). All type I keratin
genes (except K18; Waseem et al.,
1990
) are clustered on
chromosome 17q21 and type II genes on 12q13 (International Human Genome
Sequencing Consortium, 2001
).
Transcription analysis has demonstrated that the diversity of keratins is not
increased further by alternative splicing.
Knowledge of IF genes and expression patterns stimulated the discovery of
point mutations in a still growing number of IF genes, which has provided
evidence for their pathogenic relevance in human disorders (Bonifas et al.,
1991; Coulombe et al.,
1991
; Lane et al.,
1992
; reviewed by Irvine and
McLean, 1999
). Such
`experiments of nature' have demonstrated that mutations in at least 14
epidermal keratin genes cause fragility syndromes of epidermis and its
appendages that seem to result from a collapse of a mutant keratin
cytoskeleton. Formally, this was the genetic proof for a true cytoskeletal
function of these proteins. Desmin mutations analogous to those in epidermal
keratins were connected to myopathies of skeletal and heart muscle (Goldfarb
et al., 1998
), whereas point
mutations in GFAP are now known to cause Alexander's disease (Brenner et al.,
2001
). At least two reports
have linked NF-L mutations to Charcot-Marie-Tooth disease type 2E (Mersiyanova
et al., 2000
; De Jonghe et
al., 2001
). Finally, mutations
in the genes coding for the nuclear lamins A/C give rise to several
tissue-restricted disorders termed laminopathies (for a recent discussion, see
Hutchison et al., 2001
; Wilson
et al., 2001
). These data
support the view that IF proteins also serve non-cytoskeletal functions
(Quinlan et al., 2001; Wilson et al.,
2001
).
Additional insight into IF protein function comes from genetically altered
mice (H. Herrmann et al., unpublished). One common theme that emerges from
such studies is that there are essential and nonessential IF protein functions
depending on the tissue context. Ablation of keratins leads to extensive
tissue fragility in the basal but not in the suprabasal epidermis (Lloyd et
al., 1995; Peters et al.,
2001
; Reichelt et al.,
2001
). Moreover, knockout
studies have demonstrated that certain IF proteins compensate each other
(Magin et al., 2000
). In
addition, the phenotype of some IF gene knockout mice has shed light on new
pathologies (Ku et al., 1999
;
Caulin et al., 2000
; Hesse et
al., 2000
; Tamai et al.,
2000
).
The analysis of diseases with IF involvement as well as the understanding
of IF function and evolution will be aided by the knowledge of the
corresponding genes. Given that currently about 40 functional keratin genes
had been identified, we were surprised by the large number of keratin genes in
the recently published draft of the human genome. To clarify whether 111
keratin genes exist in the human genome (International Human Genome Sequencing
Consortium, 2001), we have set
out to analyze the data-set available in the public domain.
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RESULTS |
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The gene density in the two keratin clusters appears much higher than
estimated for the overall genome and is approximately 35 kb per gene. There
are 111 pseudogenes plus 47 gene fragments for all keratins. Intron-containing
pseudogenes are mostly contained within the two keratin clusters, whereas
those with features of processed pseudogenes have invaded most chromosomes,
often at several positions (Fig.
2). A few earlier analyses have identified pseudogenes for
keratins 8, 14, 16, 17, 18, 19 and hair keratins (Kulesh and Oshima,
1988; Rosenberg et al.,
1988
; Waseem et al.,
1990
; Troyanovsky et al.,
1992
; Ruud et al.,
1999
; Smith et al.,
1999
; Hut et al.,
2000
; Rogers et al.,
2000
; Winter et al.,
2001
). The peudogenes coding
for K14, K16 and K17, which arose by gene duplication, are located outside the
type I keratin cluster.
|
Unexpectedly, processed pseudogenes, which are cDNA derivatives, show a
strikingly uneven gene relatedness. By far the highest number of processed
pseudogenes relates to keratin genes 8 and 18, which map adjacently on
chromosome 12q13 within the type II gene cluster. K8 and K18 are typical of
internal epithelia and represent the earliest intermediate filament expression
pair in embryogenesis. There are 62 processed pseudogenes plus 15 gene
fragments for the keratin 18 gene, and 35 processed pseudogenes plus 26 gene
fragments for the keratin 8 gene (for a previous notion of pseudogenes, see
Kulesh and Oshima, 1988;
Waseem et al., 1990
). These
processed pseudogenes are dispersed over all chromosomes (see
Fig. 2). None of these
pseudogenes contained an intact open reading frame. Other keratin genes are
either true single copy genes or are accompanied by one to four pseudogenes
(Fig. 1).
In the present draft, no gene for keratin 11 (Moll et al.,
1982), which may represent a
polymorphic variant of K10 (Mischke and Wild,
1987
; Korge et al.,
1992
) or for K6c-f (Takahashi
et al., 1995
) were found. The
status of the latter may have to await the completion of the human genome.
Novel keratin genes and nomenclature
We discovered seven new type II keratins. Of these, five displayed homology
to K6a, K6b and K5, one was most closely related to K1 and one was highly
similar to K6b (Fig. 3). This
new member of the K6 family has 99% protein sequence identity to K6b, but at
the genomic level it contains a completely different intron 3. The
evolutionary relationship of keratins is outlined in
Fig. 4. Owing to the incomplete
alignment of contigs, a few additional keratin genes and pseudogenes may
exist.
|
|
The total number of keratin genes amounts to 49. Our survey of the current draft of the human genome conforms well with the view of 22 keratins expressed in various epithelia, 15 trichocyte-specific, 5 inner root sheath and 7 novel keratins described in this report. Together with the 13 genes for the non-keratin IF proteins, the number of genes encoding cytoplasmic IF proteins reaches 62. The three nuclear lamin genes bring the entire IF multigene family to 65.
Based on the numbering system introduced by Moll and colleagues (Moll et
al., 1982), we propose to name
novel type II keratins according to their sequence relationship with one of
the existing eight type II genes, followed by a small letter. The type II
keratin genes reported in this study are therefore named K1b, K5b, K5c, K6h,
K6i, K6k and K6l. Type I keratins should be named in the same way (see also
Fig. 1). Novel genes not
related to existing proteins should be given new numbers starting with
K21.
Non-keratin IF genes
All 13 genes encoding the non-keratin cytoplasmic IF proteins are covered
by the draft sequence (Fig. 1).
Given the considerable sequence drift among these genes, the chicken sequence
of synemin was non-informative for the identification of human synemin. The
human orthologue was identified by D. Paulin (M. Titeux et al., unpublished).
No additional functional IF gene was recognized in the current draft.
Interestingly, pseudogenes are very rare among the non-keratin genes. Only the
neurofilament NF-H gene is accompanied by two pseudogenes. Also, the genes for
the three nuclear lamins (lamins A/C, B1 and B2) lack pseudogenes. If the
completed version of the human genome lacks an additional lamin gene, the
oocyte-specific lamin of certain amphibia
(Döring and Stick,
1990) has no orthologue in the
human genome.
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CONCLUSIONS AND PERSPECTIVES |
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In view of the well-conserved structure of IF proteins and the common
principles governing their assembly properties, a search for mutations in
known and newly discovered IF protein genes is likely to reveal their
involvement in additional disorders and to unravel new IF functions (see also
Quinlan, 2001).
Most vertebrate gene families have pseudogenes, but these usually represent
only a small minority of the total gene number (Mighell et al.,
2000). Thus, the large number
of pseudogenes for the keratin gene family is startling. Particularly striking
is the finding that some 87% of these pseudogenes relate to keratin genes 8
and 18. An uneven distribution also holds for the human actin pseudogenes.
There are 23 pseudogenes for ß- and 6 for
-cytoplasmic actin,
while the four muscle actin genes lack pseudogenes (Pollard,
2001
). The molecular
mechanisms resulting in the generation of pseudogenes from some but not other
genes are unknown. However, a future analysis of their integration sites may
yield further information about the structural properties of human chromatin
and the mechanisms of recombination.
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ACKNOWLEDGMENTS |
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Footnotes |
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While this manuscript was under review, Mizuno et al. characterized
desmuslin, an IF protein that interacts with -dystrobrevin and desmin
(Mizuno et al., 2001
). When we
compared its sequence with that of human synemin, we found it to be nearly
identical to the synemin
splice variant described by M. Titeux et al.
(unpublished). Therefore, we propose to use the established name synemin.
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