(Received for publication, December 20, 1995)
From the
In both mammals and birds, the creatine kinase (CK) family
consists of four types of genes: cytosolic brain type (B-CK); cytosolic
muscle type (M-CK); mitochondrial ubiquitous, acidic type
(Mi-CK); and mitochondrial sarcomeric, basic type
(Mi
-CK). We report here the cloning of the chicken
Mi
-CK cDNA and its gene. Amino acid sequences of the mature
chicken Mi-CK proteins show about 90% identity to the homologous
mammalian isoforms. The leader peptides, however, which are
isoenzyme-specifically conserved among the mammalian Mi-CKs, are quite
different in the chicken with amino acid identity values compared with
the mammalian leader peptides of 38.5-51.3%.
The chicken
Mi-CK gene spans about 7.6 kilobases and contains 9 exons.
The region around exon 1 shows a peculiar base composition, with more
than 80% GC, and has the characteristics of a CpG island. The upstream
sequences lack TATA or CCAAT boxes and display further properties of
housekeeping genes. Several transcription factor binding sites known
from mammalian Mi-CK genes are absent from the chicken gene. Although
the promoter structure suggests a ubiquitous range of expression,
analysis of Mi
-CK transcripts in chicken tissues shows a
restricted pattern and therefore does not fulfill all criteria of a
housekeeping enzyme.
A sufficient capacity and balanced regulation of ``high
energy phosphate'' supply and turnover is essential for the proper
function of any cell. Large amounts of energy-rich phosphagens can be
found in many cells or tissues throughout the animal kingdom. In all
vertebrates and also in some invertebrates this phosphagen is
phosphorylcreatine (PCr)()(1) . PCr and ADP are the
products of the reversible transfer of
-phosphate groups from ATP
to creatine, catalyzed by the creatine kinases (CKs). Two fundamental
types of CKs can be found in vertebrates: cytosolic and mitochondrial
CKs. The subcellular localization, the biochemical and kinetic data,
and the loss of flagellar motility in spermatozoa upon inhibition of
the CK system and other data (for review, see (2) ) led to the
suggestion of a metabolic PCr circuit with PCr as a transport and
storage form of high energy phosphate. The PCr circuit connects sites
of high energy phosphate production (glycolysis and oxidative
phosphorylation) with those of high energy phosphate consumption. At
the producing end of the circuit, CK is thought to have privileged
access to ATP generated either by glycoclysis or by oxidative
phosphorylation in the mitochondrial matrix and uses this ATP to
generate PCr. At the receiving end, CK is functionally coupled to
various ATPases (for instance myosin ATPase of myofibrils), which use
the ATP generated in the reverse CK reaction.
In chicken there are
five different CK subunits known so far; three are found in the
cytosol, two in mitochondria. The cytosolic subunits are called M-CK
(muscle) and B-CK and B
-CK (brain, more acidic
and more basic, respectively) and can dimerize with each
other(3, 4, 5) . They are found soluble in
the cytosol, but fractions are also associated, for instance, with the
M-line of the sarcomeres (6) , the sarcoplasmatic
Ca
-ATPase(7) , or the spermatozoan tail. (
)Whereas in mammals there is just one isoform of B-CK, the
two B-CK isoforms of the chicken are derived from a single gene by
alternative splicing of the second exon(9, 10) .
Additional heterogeneity of B-CK was shown to be due to alternative
initiation of translation (11) or posttranslational
phosphorylation(12, 13, 14) . In tissues of
adult chicken, M-CK is predominantly found in skeletal muscle, but
contrary to mammals, it is absent from heart(3) . B-CK is
expressed in almost all tissues and found enriched in various regions
of the brain, retina, heart, gizzard, gut, and sperm(2) .
Evidence for two different isoforms of chicken mitochondrial CK
(Mi-CK) has been found by comparison of translated cDNA sequences,
isolated from a leg muscle cDNA library, to partial amino-terminal
protein sequences of Mi-CK purified from brain(15) . These two
isoforms were termed Mi-CK (a = more acidic pI, in
mammals it was called ubiquitous Mi-CK by other authors) and
Mi
-CK (b = more basic pI, sarcomeric Mi-CK in
mammals) and are found in vivo exclusively as homodimers and
homooctamers(16, 17) . The Mi-CKs are synthesized in
the cytosol as precursor proteins with distinct leader peptides (15, 18, 19) and get imported into the
intermembrane space of mitochondria. Mi
-CK is normally
coexpressed with M-CK and is present in skeletal muscle and
heart(15) , but it also has been found in sperm.
Mi
-CK is distributed more ubiquitously and has been
detected, like B-CK, in chicken brain, gut, and retina. Hence the
expression of the mitochondrial CKs matches to a certain degree the
pattern found for the cytosolic CKs, indicating possible common
regulatory mechanisms.
Recently a comparison of the 26 known CK protein sequences suggested a highly conserved protein family and allowed the construction of an evolutionary tree(20) . This tree predicts three gene duplications at the origin of the four CK isoforms, which is in agreement with observations on the gene structures of the four published human CK genes(21, 22, 23, 24, 25) . In chicken, only the B-CK gene has been completely analyzed so far, and fragments of the M-CK gene indicate an extraordinarily big size(10) .
To gain further insight into the evolution of the
CK isoenzyme family, the chicken Mi-CK protein sequence and
the Mi
-CK gene structure were elucidated as the first
nonmammalian Mi-CK gene. The chicken Mi
-CK leader peptides
were analyzed in order to determine if the functional chicken peptides
are isoprotein-specifically conserved. Analysis of the promoter
elements of the Mi
-CK gene was carried out to investigate
if the expression pattern of Mi
-CK is coupled to B-CK
expression, indicating common regulatory mechanisms of these two genes.
In order to test if such mechanisms are active in the chicken, the
expression patterns in several adult tissues were studied. The
structure of the chicken Mi
-CK gene, including the
localization of intron-exon boundaries was analyzed, and the structure
was compared with the mammalian ubiquitous Mi-CK genes, allowing
conclusions on the evolutionary relationship of these genes.
The whole amplified fragment, labeled with P, was used to screen a chicken brain
gt10 cDNA
library (Stratagene). A total of 2.5
10
plaques was
screened at a density of 25.000 plaques/plate (100
100 mm).
Lift-offs were performed with Biodyne type A nylon membranes (1.2
µm, Pall Inc., East Hills, NY) according to the
manufacturer's conditions and were hybridized as described
previously (30) without formamide at 68 °C overnight.
Washing was done at room temperature with three changes of 2
SSC, 0.1% SDS, 15 min each.
A genomic EMBL3 library with high
molecular weight DNA from chicken liver (Clonetech, Palo Alto, CA),
partially digested with MboI, was obtained from Dr. B. Trueb
(Maurice E. Müller Institute, Berne, Switzerland).
A total of 3.6 10
plaques was screened at a density
of 10
plaques/plate. Plaque lift-offs were done as
described above. Hybridization with the
P-labeled fragment
of clone UB15-9 (EcoRI/SmaI fragment,
nucleotides 170-487 (see Fig. 1A)) was done as
above, but in the presence of formamide at 42 °C. Lift-offs were
washed with 2
SSC, 0.1% SDS at 65 °C.
Figure 1:
Structure and
partial restriction map of the chicken Mi-CK gene locus, of
genomic
clones and of the derived cDNA. A, the
nucleotide sequence of chicken Mi
-CK cDNA is represented as
a bar. The intron-exon boundaries are shown as vertical
lines, and the vertical numbers above represent the
5`-ends of the exons. The 5`- and 3`-UTRs are represented by open
boxes, and the part coding for the leader peptide is hatched. The filled black box outlines the exon that
is conserved in all CK genes (see ``Discussion''). The
fragment amplified in RT-PCR (UB11-83) is shown with the two
degenerate oligonucleotides (boldface), used in the final
amplification (also see ``Materials and Methods''). The cDNA
fragment isolated by library screening (UB15-9) and the extension
in the direction of the 5`-end obtained by RACE-RT-PCR (TW61-11)
are indicated as well. B, the organization of the chicken
Mi
-CK gene locus was derived from the analysis of three
(gMia-64, -67, -72) of the six genomic
clones. The localization
of these three clones along the gene locus is indicated. Clone 72 is
rearranged at a Sau3AI site in intron 7 as verified by direct
sequencing of the
clone. BamHI fragments of clone 72
were subcloned and sequenced in part (see sequencing strategy,
indicated by arrows). The region of the BamHI sites
and of the 3`-end of the gene were sequenced directly on the
corresponding genomic
clones (indicated by thick bars above the lines of the
clones). The sequencing strategy is
shown by arrows, and intron 1 has not been sequenced in total
as indicated. At the ends of the genomic
-clones there are BamHI sites that probably are cloning artifacts from a MboI-BamHI fusion. At the 5`-end of clone 67 is an
additional BamHI site. Because clone 72 contains a upstream HindIII site, which in combination with the HindIII
site in clones 64 and 67 gives rise to the same HindIII
fragment as the one found in genomic Southern blots, it represents a
faithful segment of the chicken Mi
-CK locus. B, BamHI; Sa, Sau3AI; H, HindIII; T, TaqI;
, parts sequenced directly on the
clones;
, 5`-UTR with length not exactly
known;
, leader peptide;
, normal exon;
, exon 6, conserved in all mammalian and
avian CKs;
,
3`-UTR.
Obtained products were verified with an
internal, P-labeled oligonucleotide by Southern blotting,
subcloning, and sequencing. Additional Mi
-CK
5`-RACE-RT-PCRs were performed with a set of oligonucleotides derived
from a 60-bp stretch located in exon 1, with methylmercuric
hydroxide-denatured RNA and with other reverse transcriptases.
All plasmids were grown in Escherichia coli XL-1 blue (Stratagene), and DNA was isolated either by an alkali lysis-type method (30) or by column preparation (Nucleobond AX kit, Machery-Nagel, Oensingen, Switzerland) according to the manufacturer's descriptions.
Plasmids pStM5 and
pStM6 were linearized with HindIII/NdeI and BamHI/EcoRI, respectively. Both were transcribed with
T7 RNA polymerase (Promega), producing probes of approximately 160 and
220 nucleotides, respectively. [P]rCTP-labeled
cRNA-probes were synthesized as follows. Three µl of 5
transcription buffer (Promega) dithiothreitol (nuclease-free, Sigma) to
a concentration of 10 mM; rATP, rGTP, and rUTP (Promega; final
concentration, 400 µM each); and 60 µCi of
[
P]rCTP were mixed in a siliconized Eppendorf
tube. Diethyl pyrocarbonate (Sigma) -treated distilled H
O
was added to give a total volume of 15 µl. Linearized plasmid
template to a final concentration of 40 to 50 ng/µl, RNAsin (20
units, Promega), and T7-RNA polymerase (20 units, Boehringer, Mannheim)
were added at the end. Transcription was done for 1 h at 37 °C. The
cRNAs were purified by a preparative acrylamide gel and recovered from
the gel slices by incubation in cracking buffer (0.3 M NaCl,
0.1 mM EDTA, 10 mM Tris, pH 7.5) for 1 h at room
temperature and subsequent extraction and precipitation.
The RNase
protection was done with the RNase protection kit from Boehringer
Mannheim. 10 µg of total RNA was used from each tissue with
10
cpm of labeled probe, and hybridization was done
overnight at 50 °C. RNase digestion was done with RNase A and RNase
T1 at 37 °C for 1 h. One-half of the assays was analyzed on a 4%
sequencing gel.
In vitro transcription in the presence of [S]UTP
(>1000 Ci mmol
, Amersham Corp.) was done
essentially as described above; 60 µCi of
[
S]rUTP were used instead of rCTP. The final
volume was 20 µl. After the first hour of incubation, another
aliquot of RNA polymerase was added, and the mixture was incubated for
another hour. The template was digested with DNase I to stop the
reaction. All probes were purified by Sephadex G-50 columns, and sizes
were controlled on an analytical 4% polyacryamide-urea gel.
Some
conditions of prehybridization, hybridization, and subsequent washing (38, 39) were slightly modified. Probe for
hybridization was diluted in hybridization buffer to a concentration of
20,000 cpm µl
, and 30 µl were applied
per slide. Dithiothreitol concentration in the hybridization buffer was
increased to 100 mM(40) . Stringent washes were
carried out at 55 °C in 0.5
SSC, 50% formamide, 10 mM dithiothreitol for 2 h.
A chicken brain
gt10 cDNA library was screened with this fragment to isolate the
full-length cDNA. Out of this screening, one clone, containing
sequences coding for Mi
-CK, was purified to homogeneity,
subcloned, and sequenced (UB15-9). This cDNA sequence starts only
170 bp downstream of the ATG initiation codon (see Fig. 1and Fig. 2), reaches to the 3`-end at 1383 bp of the mRNA (see Fig. 1A), but contains no poly(A) tail. The RACE-RT-PCR
method (31) is a way to amplify unknown cDNA ends, with the
advantage of avoiding time-consuming screening procedures. Hence this
method was used to verify the 3`-end of the chicken Mi
-CK
cDNA. In this experiment, a poly(A) tail was found at the expected
position. The polyadenylation signal sequence CATAAA was identified 19
bp upstream from the 3`-end (see Fig. 2, boxed nucleotides at the end). The 3`-UTR is 134 bp long, but it lacks the sequence
similarities observed by others (42) in the 3`-UTRs of human,
rat, and mouse Mi
-CKs. For the chicken Mi
-CK, a
message size of approximately 1700 bp is observed in Northern blot
experiments (not shown). Since poly(A) tails usually range between 100
and 200 bp, the Mi
-CK cDNA should have a length in the
range of 1500 bp. Hence the missing sequence at the 5`-end might be
250-350 bp long.
Figure 2:
Partial sequence of the chicken
Mi-CK gene. The sequences were determined as indicated in Fig. 1(arrows). The length of intron 1, which was not
sequenced in total, was determined by analysis of restriction enzyme
digests, and only part of the 1 kb that was sequenced is shown. Capital letters represent exon sequences, whereas lower
case letters stand for intron and 3`- and 5`-flanking sequences of
the gene. All intron-exon boundaries were sequenced, and splice sites
were identified in all cases (underlined; points refer to
nucleotides that do not match the consensus splice sequences). The
polyadenylation signal is boxed. In the promoter region, the
two GC boxes are underlined twice (&cjs0808;), and the
putative AP-2 binding sites are indicated by a single line above the sequence. The two potential E2A contact sites are
shown by arrows below the sequence and the MRE-site
by an arrow above. The derived amino acid sequence is
indicated under the respective exon, and the mitochondrial import
signal is underlined. The numbering of the protein sequence
starts with the first amino acid of the mature protein. Thus the
initiating methionine is amino acid -39. The nucleotide sequence
is not numbered with the exception of the region upstream of the ATG.
There are 120 nucleotides/full line, and every 10 nucleotides there are asterisks on top of the figure.
The RACE-RT-PCR method was also applied to
amplify these missing 5`-terminal sequences. The largest extension
obtained from these experiments provided only 83 additional bp compared
with the cDNA clone UB15-9 (Fig. 1A). Other
attempts, using strongly denaturing agents like methylmercuric
hydroxide to melt possible secondary structure elements in the RNA, did
not yield any further sequence information. The method was working in
our hands, as the missing 5`-end of chicken Mi-CK (15) was cloned at the same time. There we obtained the
remaining 57 bp of the coding region, and the 156 bp of the 5`-UTR (the
complete sequence of the Mi
-CK leader peptide is given in Fig. 5). Thus at the 5`-end of chicken Mi
-CK,
200-300 bp resisted cloning by cDNA-based methods for unknown
reasons.
Figure 5:
Mi-CK mRNA is not accumulated
in all tissues of adult chicken expressing B-CK. RNase-protection
experiments with
P-labeled cRNA-probes derived from the
3`-regions of B-CK (A) and Mi
-CK (B) were
performed as described under ``Materials and Methods.'' 5
µg of total RNA from brain(2) , leg muscle(3) ,
heart(4) , gut(5) , kidney(6) , and testis (7) was used per lane. In lane 1, the undigested probe
was loaded (specific activity of the B-CK probe > 4.6
10
cpm/µg; specific activity of the Mi
-CK
probe > 6.3
10
cpm/µg). The autoradiographs
for B-CK and for Mi
-CK were exposed for 12 h at room
temperature, except for the testis lane of section B, which is
from a 24-h exposure. u, undigested; p,
protected.
The missing sequences were finally obtained by analysis of
genomic clones (see below). They code for the leader peptide of
39 amino acids, necessary for import into mitochondria, and the 5`-UTR,
which we predict to have length of
100 bp. The whole cDNA without
poly(A) tail has therefore a length around 1480 bp.
Figure 3:
Comparison of the leader peptides of the
known Mi-CKs. A, the leader sequences of the known
Mi-CKs (or ubiquitous Mi-CKs) and Mi
-CKs (or
sarcomeric Mi-CKs) were aligned and arranged using the programs Pileup
and Pretty of the GCG software package(45) . An amino acid was
put into the consensus and replaced by a hyphen in the corresponding
sequence if it was identical in six of the seven sequences. B,
percentages of amino acid sequence identity. The upper right half shows the identities within the leader peptides, whereas the lower left half shows those within mature proteins. Humubi, human ubiquitous; Ratubi, rat ubiquitous; Mouubi, mouse ubiquitous; Ch, chicken; Humsar, human sarcomeric; Ratsar, rat
sarcomeric.
The amino acids conserved in all of the leader peptides of
either the Mi-CK or the Mi
-CK are, with one
exception, also conserved in the chicken Mi
- and
Mi
-CKs. Preliminary expression studies done with the
chicken Mi
-CK cDNA have shown that the Mi
-CK
leader peptide is functional and that Mi
-CK gets imported
into mitochondria of heterologous mammalian CV-1 cells. (
)
In total, close to 5.3 kb have been sequenced (Fig. 2)
including 500-600 bp of putative promoter region. About 300 bp of
promoter sequences further upstream were analyzed, but they are only
sequenced on one strand and therefore are not included. The
nonsequenced part is located in intron 1. At the 3`-end, there are 83
bp of known sequence after the polyadenylation site. The chicken
Mi-CK gene is approximately 7.6 kb long and has nine exons.
The defined exon sizes range from 86-247 bp, but the putative
exon 1 may be larger. Intron 1 is 4.2 kb long, of which 1.2 kb were
sequenced. All other introns are rather small (<520 bp), giving rise
to a compact gene structure on the 3`-side. Analysis of the nucleotide
sequences around the intron-exon junctions (underlined in Fig. 2) shows that they correspond well to the AG-GT splice
junction rule (44) for intron-exon boundaries. The dots in the
splice regions indicate deviations from the rule.
The 5`-region of
the chicken Mi-CK gene shows a high GC-content. It is
around 80% in the whole BamHI fragment comprising exon 1 (see Fig. 1B), and the same holds true for the entire exon
1, regardless of the size of 5`-UTR proposed. The three mammalian
Mi
-CKs (19, 24, 41) also have a
higher GC-content in the exon 1 region of the cDNA, but it is 15% below
the content found in chicken. Strong RNA secondary structures, as
predicted by the Stemloop program in the GCG software
package(45, 46) , are an additional feature of exon 1.
Both the high GC content and the strong secondary RNA structure
prevented the mapping of the transcription start site of the chicken
Mi
-CK gene (see Fig. 1and Fig. 2), although
we tried primer extension analysis and RNase- and S1-protection
analysis. From the putative transcription factor binding sites found
(see below and Fig. 2), a transcription start site about 100 bp
upstream of the ATG initiation codon seems most likely. Alternatively
an additional, untranslated exon might exist, but in the sequences
upstream of the ATG there was no indication of a further splice site.
Therefore it is highly probable that exon 1 is in fact the first exon
and that transcription initiates about 100 bp upstream of the ATG.
Analysis of the chicken Mi-CK gene with the Grail
software (47) predicts, around exon 1 (see Fig. 1and
2), a CpG island with a very high CpG score of 0.98 (for definition of
this score, see (48) ). The island starts at -587 bp
upstream of the ATG and extends to +506 bp downstream. The human
Mi
-CK gene also has such a CpG island from -198 to
+236 bp, with a score of 0.73. In both cases, it has to be
investigated if these islands are undermethylated, but their presence
in the promoter regions is of interest in the context of their
regulation. A lot of so-called housekeeping genes (49) or
tissue-specific genes with a broad range of expression (50) do
have such CpG islands. In addition, these genes usually lack TATAA and
CCAAT boxes. The putative promoter region of the chicken
Mi
-CK gene fulfills all of these features and is typical
for such a gene type. A search for specific potential binding sites for
transcription factors upstream from the ATG revealed, among many SP1
sites (14 on the upper strand), two GC boxes that match the consensus
defined by Kadonaga et al.(51) and are marked with a double line in Fig. 2. Further potential binding sites
were found for the general factor AP2 (single lines above the sequence) matching the consensus sequence 5`-CCCMNSSS-3` (52) for the ubiquitously expressed products of the E2A gene
(5`-GCAGGTGGC-3`, arrows below the sequence) and for
a metal response element (MRE, at -82 bp, arrow above),
which is identical to the MREa element in the mouse metallothionein-I
gene promoter(53) . Suzuki and co-workers (54) have
identified three sequence elements (Mt1, Mt3, and Mt4) common in the
5`-flanking regions of nuclear genes coding for mitochondrial proteins.
The same three stretches are also found in human sarcomeric and mouse
ubiquitous Mi-CK. However, none of these
``mitochondria-related'' binding sites is found in the
sequenced upstream region of the chicken Mi
-CK, although
they might be located further upstream. Glucocorticoid and estrogen
response elements were reported in mouse and human ubiquitous Mi-CK
genes in introns or downstream of the gene. In chicken, only some,
possibly nonfunctional, half-sites for the glucocorticoid response
element are found in intronic regions.
The same 317-bp fragment used for the screening of the genomic library was used as a probe and produced only one signal per lane on a Southern blot of DNA digested with BamHI, HindIII, and TaqI (Fig. 4A). The observed hybridizing fragments were 8.5, 11, and 2.9 kb long, respectively, and are in perfect agreement with the restriction map derived from the genomic clones as shown in Fig. 1B.
Figure 4:
There is only one Mi-CK gene
in the chicken genome. 10 µg of genomic DNA each were digested with BamHI (lane 1), HindIII (lane 2),
and TaqI (lane 3); the fragements were resolved on an
agarose-gel and transferred onto a nylon membrane. The first blot (A) was hybridized to a
P-labeled 319-bp cDNA
fragment (nucleotides 169-488 in Fig. 1B; random
prime labeling; specific activity, 10
cpm/µg) and the
second (B) to a
P-labeled 63-bp cDNA fragment
(nucleotides 88-151 in Fig. 1B; PCR labeling;
specific activity, 4
10
cpm/µg). The blots were
exposed to an x-ray film for 2 days. The fragments of the marker (a
combination of phage
DNA cut with EcoRI and HindIII and phage
DNA cut with BglI) are
indicated in kb. The hybridization pattern shows that there is a single
gene and that the genomic
clones contain hybridizing fragments of
identical size.
To further support the gene structure
reported here, another Southern blot (Fig. 4B) was
hybridized with a 64-bp probe, labeled by PCR, from the 5`-end of the
known cDNA sequences (nucleotides 88-151 of the cDNA depicted in Fig. 1A; corresponding to part of exon 1). Again the
fragments of 1.1, 11, and 2.3 kb, respectively, were the same as those
found in the genomic clones. These data therefore indicate that
the chicken Mi
-CK gene is a single copy gene. Additionally
the 317- and 64-bp probes both hybridize in HindIII-digested
DNA to a fragment of the same length of 11 kb. This shows that the
organization of the
clones correctly represents the genomic
locus.
Because it is possible that small groups of cells express
Mi-CK even in tissues where no expression in whole tissue
RNA was detected, in situ hybridizations were performed.
Paraffin sections from spinal cord, liver, gizzard, and gut were
hybridized with the same probes but labeled with
S. There
was no hybridization in liver either for B- or for Mi
-CK
mRNA (Fig. 6, A-C), and B-CK was the
only CK present in the smooth muscle portion of the gizzard (Fig. 6K).
Figure 6:
Mi-CK is not localized in
smooth muscle tissues of gut and gizzard. Mi
-CK and B-CK
mRNA was localized in liver, spinal cord, gut, and gizzard by in
situ hybridization with the same, but
S-labeled cRNA
probes as in the RNase-protection experiment (Fig. 5). Sections
were from liver (A-C), spinal cord (thoracic
vertebrae; D-F), gut (G-I), and gizzard (J-L). A, D, G, and J, bright field
micrographs; B, E, H, and K, dark
field micrographs of sections hybridized with the B-CK specific probe; C, F, I, and L, dark field
micrographs of sections hybridized with the Mi
-CK-specific
probe. In the three consecutive liver sections shown, the folds in the
tissue are due to sectioning artifacts. The folds led to false
hybridization signals because of trapping of the probe. The arrows in micrographs H and I give the position of the
muscularis mucosae. The level of Mi
-CK hybridization in the
spinal cord (F) is low on the outside of the neuronal cell
bodies, and comparison to the negative control (not shown) revealed
that it represents background. nc, neuronal cell body; dh, dorsal horn; lm, longitudinal smooth muscle; cm, circular smooth muscle; mm, muscularis mucosae; v, villi; l, lumen; sm, smooth muscle
tissue; ep, epithelial layer. The in situ hybridizations were exposed for 14 d. The bar corresponds
to 200 µm.
In gut, the signal of the B-CK probe was
localized over the longitudinal and circular smooth muscle tissue. The
B-CK probe hybridized to the base of the villi and a diminishing signal
was detected toward the luminal region. There is also hybridization in
a ring just inside the smooth muscle layer, which represents the
muscularis mucosae. The hybridization to the muscle tissue seems
stronger than in the villi. Mi-CK was restricted to villi
and showed no expression in the surrounding smooth muscle tissue (Fig. 6, G-I).
Finally, in spinal
cord, Mi-CK showed a punctuated hybridization pattern,
which localized over the cell bodies of neurons of the gray matter;
there it is expressed together with B-CK. In other cells, only
unspecific hybridization was observed. On the other hand, B-CK
hybridizes at a lower level in gray as well as in white matter (Fig. 6, D-F) and seems not to be
restricted to neuronal cells.
Recently several reports described Mi-CK amino acid sequences
and the corresponding gene structures of mammalian Mi-CK genes, and
regulatory and evolutionary aspects of the Mi-CKs were
proposed(20, 24, 25, 54) . Among the
nonmammalian species, the chicken CK isoenzyme family has already been
well documented(2, 10, 11, 57, 58, 59, 60) with
the exception of the fourth gene, Mi-CK. Here we present
the chicken Mi
-CK amino acid sequence derived from the cDNA
as well as the structure of the corresponding gene. The analysis of the
chicken system and its comparison with the mammalian CK set allows
conclusions to be drawn on functional elements of the protein, like the
leader segment, on regulatory properties of the chicken
Mi
-CK promoter leading to regulated expression, as well as
on evolution of the CK isoforms.
An amino acids sequence comparison (20) of the creatine kinase isoenzymes has shown that the
proteins can be arranged into six different groups based on their
levels of sequence identities. Two groups consist of the cytosolic CKs
of the fishes/amphibians (see ref. 20) and will not be further
discussed, while the other four groups are formed by the different
isoforms of mammals and birds. The nine exons of the
Mi-CK-gene give rise to a precursor protein of 417 amino
acids, including an amino-terminal mitochondrial import sequence of 39
amino acids. The mature chicken Mi
-CK fits in the
comparison into the group of the Mi
-CKs or ubiquitous
Mi-CKs. The levels of identities between the ubiquitous Mi-CKs known so
far are around 90% for the mature protein as shown in Fig. 3B.
In addition, an isotype-specific
conservation of the leader peptides was noted for mammalian Mi-CKs, and
hence it was suggested that these peptides might act as
isoprotein-specific import sequences or bind to specific import
receptors(19) . The observed low identities with the chicken
leader peptides (Fig. 3B) are not in favor to extend
this hypothesis also to nonmammalian isoforms, unless the
isoprotein-specifically conserved residues found between chicken and
mammalian leader peptides (amino acid positions 10, 11, 18,
23-25, 27, 28, and 30) were sufficient to ensure the specific
import (Fig. 3B). In contrast to the leader peptides,
the mature protein Mi- and Mi
-CK sequences are
conserved isoform-specifically to a degree of almost 90%, and no
segments can be found in the mature proteins where the conservation
drops to the low levels of the leader peptides. Hence, the reduced
conservation of the leader peptides cannot be explained by the greater
phylogenetic distance of the chicken from mammals since leader peptide
and mature proteins must have undergone simultaneous evolution. The
noted high homologies in the mammalian peptides are therefore a mere
fact of their phylogenetic closeness, and the apparent lower
evolutionary pressure for conservation of the leader peptides is only
observed when the large evolutionary gap, as the one between mammals
and birds, is analyzed. Thus, it is unlikely that the conservation of
the mammalian Mi-CK leader peptides represent isoform-specific
functions. This is supported by the observed import of the chicken
Mi
-CK into the mitochondria of mammalian fibroblastic CV-1
cells, where endogenous Mi
-CK, but no Mi
-CK,
can be expected.
On the level of the nucleotide sequences, 78%
identity is observed if the mature chicken Mi-CK is
compared with any of the mammalian ubiquitous Mi-CKs. As conservation
on the protein level with 90% is much higher, nucleotide changes occur
therefore mainly at wobble positions. The same holds true for the
Mi
-CKs or any of the cytosolic CKs. In case of the UTRs,
the situation is different. Cheng et al.(42) have
shown that a sequence in the 3`-UTR of the rat ubiquitous Mi-CK is
involved in regulation of its expression/translation. Whereas this
sequence stretch of 72 bp is conserved in the mouse and the human
sequence, it cannot be found in chicken Mi
-CK, suggesting
that this mechanism of regulation is not active in chicken.
A
schematic representation comparing the four known Mi-CK genes is given
in Fig. 7. The organization of the chicken gene is similar to
the two other known Mi-CK genes from man and mouse. The
localization of intron-exon boundaries in the coding region of the cDNA
and the exon-sizes (except number 1) are conserved between these genes.
In addition, the chicken gene lacks a noncoding first exon, like the
mammalian ubiquitous Mi-CKs but contrary to all other known CK genes.
Whereas the mouse and the human genes show a common bipartite gene
structure with a clustering into the goups of exons 1-6 and
7-9(41) , in chicken Mi
-CK, exon 1 is
separated from the compact rest of the gene by a rather large intron.
The sarcomeric Mi-CK gene, although it is with 37 kb the largest Mi-CK
gene known so far and in addition has two noncoding first exons, has
exon intron boundaries at the same positions of the coding region as
the ones noticed in the ubiquitous Mi-CK genes (see Fig. 7).
Hence, the two gene types might have evolved by a gene duplication
event.
Figure 7:
Conservation of the Mi-CK gene structures
in mammals and birds. The chicken Mi-CK gene structure is
compared with the three other known Mi-CK genes of human and mouse (24, 25, 41) . The exon sizes are not drawn
to scale, but for any given gene, they are proportional in size. The
coloring of exons is as mentioned in Fig. 1. The sizes of the
genes are indicated below the names in kb; due to its large size, the
scale of human sarcomeric Mi-CK is much smaller as indicated by the scale bar. The numbers below the chicken gene refer to the
nucleotide positions of the intron exon boundaries in the chicken
Mi
-CK cDNA. As the mammalian Mi-CKs have one amino acid
more in their exon 1, the corresponding numbers have to be increased by
three (153, 352, and so on). The numbers to the right indicate the
3`-end of the cDNAs without the poly(A) tail.
, 5`-UTR and 3`-UTR of known length;
, heterogenous transcription start site;
, 5`-UTR length not exactly known;
, coding region of leader peptide;
, normal exon;
, exon 6, resp. 8 conserved in all
mammalian and cytosolic CKs; sar, sarcomeric Mi-CK; ubi, ubiquitous Mi-CK; Mi
-CK, acidic isoform of chicken
Mi-CK.
As noted earlier (25) the mitochondrial CK gene structure is different from that of the cytosolic CKs. There is only one exon (exon 6, respectively exon 8 in human sarcomeric Mi-CK) that is conserved through all of the CKs in its size and localization in the coding region (black exon in Fig. 7). Other features, for instance the conserved 5`-noncoding exon in case of the cytosolic CK genes, are not found in the Mi-CK genes. Interestingly the region of the conserved exon is also the exon with the highest homology at the amino acid level among the different guanidino kinases(20) . This might indicate a critical role of these residues for the structure and function of guanidino kinases, as has already been shown for a tryptophane residue in this region(62) .
All of the data mentioned so far show that the CKs form a group of evolutionarily related isoenzymes and can further be embedded into the larger family of the guanidino kinases. The four different CK isoforms most probably evolved by three gene duplication events, with the first of these producing a primordial cytosolic and a primordial mitochondrial isoform. The phylogenetic tree, which can be derived from the protein comparison(20) , suggests this first duplication to have occurred before the separation of the echinoderms from chordates, which is in agreement with published data on the expression of Mi-CKs in sea urchins(63, 64) . The significant homology, still found if the protein comparison of the CKs is extended to other guanidino kinases like ArgK or guanidinoacetate kinase, is a strong indication for a common ancestor of the guanidino kinases. ArgK has been suggested to be this ancestor on the basis of data on its dimerization capacity with cytosolic CKs, the nature of its substrate arginine as being part of basic metabolism, and especially from the distribution of arginine and ArgK in the animal kingdom(1) . However, creatine has been found in sperm of many nonvertebrate taxa as well(1, 65) . Taking into account that the recently cloned guanidinoacetate kinase shows higher homology to CKs than to ArgKs(56) , it can be suggested that a duplication event has first produced a guanidinoacetate kinase and ArgK and that later CK has evolved from guanidinoacetate kinase.
The promoter region of chicken
Mi-CK is rather similar to those of the human and mouse
genes and displays all of the features attributed to housekeeping genes (49) or tissue-specific genes with a broad range of
expression(50) . It is at present not known whether any of the
transcription factor binding sites identified by sequence analysis are
actually used for the regulation of the chicken Mi
-CK gene.
During rat pregnancy, B-CK as well as ubiquitous Mi-CK
(Mi
-CK), are regulated in rat uterus by steroid hormones,
and binding sites for steroid receptors are expected. However, in the
chicken Mi
-CK gene, no such sites have been identified, and
only possibly nonfunctional half-sites for glucocorticoid receptors
have been found. On the other hand, it has been shown for rat B-CK that
the regulation is independent of the binding of an estrogen receptor to
the promoter region(66) . The three binding sites Mt1, Mt3, and
Mt4 (54) identified in nuclear genes coding for mitochondrial
proteins have also been found in the mouse ubiquitous Mi-CK gene. These
sites are present neither in the chicken nor the human gene, but in
both cases the known 5`-located sequences may be to short to contain
these sequences. Summarizing the data shown, the Mi
-CK gene
displays in part the same regulatory elements found in mouse and human.
Some of the additional elements reported especially in mouse are not
found in the chicken Mi
-CK gene, probably due to the
limited sequence information at the 5`-end. The ``missing''
binding sites might, however, be important to explain the observed
restricted expression pattern found for chicken Mi
-CK.
The presence of Mi-CK in brain tissues is already well
documented(2, 60) . Our localization in spinal cord
shows that Mi
-CK is coexpressed with B-CK in the cell
bodies of neural cells of the gray matter but is absent from any other
region where only B-CK is found. Hence, neurons seem to rely on a
functional PCr shuttle in spinal cord. Whereas other investigators
reported minute CK expression in liver, our in situ hybridizations show no Mi
-CK or B-CK expression in
general. Either the transcripts are below detection limit or not
present at all. The smooth muscle-containing tissues analyzed by in
situ hybridization do not express Mi
-CK in their
smooth muscle portions, but they display considerable amounts of B-CK.
Hence, gut (duodenum) and gizzard smooth muscle in chicken function in
the absence of Mi-CK, which is different from vascular and intestinal
smooth muscle of guinea pig (8) or smooth muscle from
rat(42) . For chicken gizzard, the lack of Mi
-CK is
due to its peculiar contractile properties (60) . Whether this
holds true for chicken gut as well is not known. The only portion in
gut expressing Mi
-CK is the border region of the villi,
which suggests that a functional PCr circuit might be important for
cells of the brush border involved in resorption processes. These data
show that Mi
-CK expression in tissues of adult chicken is
more restricted than that of B-CK. They indicate that the name
ubiquitous, given to the mammalian Mi
-CKs, is not
justifiable in chicken. The features of the Mi
-CK promoter
indicating a housekeeping gene are misleading, and there must be other
regulatory elements narrowing its expression. The putative additional
regulatory elements will have to determined by future research.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X96402 [GenBank]and X96403[GenBank].