RESULTS Molecular genomic cloning of the R.marmoratus Hsc71 (rm-Hsc71m) To examine heat shock gene expression in Rivulus, we isolated hsp70-related genes from a Rivulus genomic library
( 30). Rivulus genomic clones
were screened by low stringency hybridization using a human hsp70 cDNA
as a probe. Seventy-six clones were isolated in the primary screen
and were placed into three groups depending on the intensity of
the hybridization signal. Restriction enzyme analysis and Southern
blotting of the strong signal group identified six clones with similar patterns
(data not shown). One was sequenced, and contained sequences encoding
the R.marmoratus hsc71 gene, which showed
high muscle-specific expression (see below); hence it was designated rm-hsc71m. To characterize rm-hsc71m, we sequenced 11
041 nt of the 13.8 kb insert DNA. As indicated in the sequence deposited
in GenBank (accession no. AF227986), the clone contained one putative
gene composed of eight exons capable of encoding 655 amino acids
and ~4.4 kb of upstream sequences. A homology search of this protein
using the Blast program revealed its high homology to the cytosolic
and constitutively expressed Hsc70 family (data not shown). Similar
to other cytosolic and constitutively expressed Hsc70 family genes, rm-hsc71m has one non-coding exon at 1704 bp upstream
of the first coding exon. A 690 bp RT–PCR product was obtained when
total RNA was subjected to RT–PCR analysis with a primer
in the putative non-coding exon and a primer in the third coding
exon (Fig. 1A and data not shown). The splice junction
of the first non-coding exon was confirmed by direct sequencing
of the RT–PCR products (data not shown). As predicted,
primer extension analysis showed that the transcription start site
was 68 bp upstream of the splice donor of the non-coding exon I
(data not shown). Thus, the rm-hsc71m gene is composed
of nine exons including the first non-coding exon and eight introns
(Fig. 1B). It is noteworthy that the first intron
of fish is much longer than the first introns seen in mammals, and
the fourth intron of Rivulus (1580 bp) is the longest among the
known species. ![Figure 1 Figure 1](picrender.fcgi?artid=55811&blobname=gke43201a.gif) ![Figure 1 Figure 1](picrender.fcgi?artid=55811&blobname=gke43201b.gif) | Figure 1 Genomic cloning of the R.marmoratus hsc71 gene. (A)
Schematic representation of the restriction map and gene structure
of the R.marmoratus hsc71 gene
(rm-hsc71m). The open box represents the untranslated
exon, whereas the filled boxes are the protein-coding (more ...) |
External stress dependency of the rm-hsc71m gene expression To determine whether rm-hsc71m is induced by
external stress, we examined its temporal expression pattern in
response to stress in 3-month-old fish by RT–PCR. Primer
sequences for RT–PCR analysis were designed to
detect selectively rm-hsc71m transcripts.
One primer contains sequences from +17 to +36
in the initial non-coding exon I which is not conserved at all,
whereas other primers in exon II or IV is one of the least conserved
sequences among the known Hsc70 families in other species (see Materials
and Methods, and Discussion). Indeed, these primers were unique
to show a single cDNA product in RT–PCR of rivulus total
RNA (data not shown). For induction by either pH-shock or heat-shock, rivulus
were reared in water at pH 4.5 for 0.5, 1 or 2 h, or in water temperatures
of 25, 38 or 40°C for 1 h, respectively. Under
these conditions expression of rm-hsc71m was unchanged
although the basal levels of rm-hsc71m expression varied
among tissues examined (data not shown). Similar results were obtained
in experiments using fish of different ages (data not shown) indicating
that expression of rm-hsc71m is not induced by
acute external stress. rm-hsc71m expression during developmental
stages To examine how rm-hsc71m expression is regulated
in early development, total RNA was prepared from unfertilized eggs through
juvenile stage larvae, and the steady state levels of rm-hsc71m transcripts
were analyzed by RT–PCR. As shown in Figure 2A, the expression of rm-hsc71m was
apparent in 6-day-old embryos (lane 4), and the highest
expression was seen in 10-day-old embryos (lane 8). It is possible
that inability to observe rm-hsc71m expression
in younger embryos is due to cell- or tissue-specific expression
of low but undetectable levels of rm-hsc71m. To
examine cell- or tissue-specific expression of rm-hsc71m,
whole-mount in situ hybridization assay was performed.
RNA probes were prepared as described in Materials and Methods.
Whole-mount in situ hybridization showed rm-hsc71m expression
in regions of the head and in somites of 3-day-old embryos (Fig. 2B, panel a). Similarly, strong expression of rm-hsc71 was apparent in the entire body in 13-day-old
embryos and comparable signals were not seen using a sense RNA probe
(compare panels b and c in Fig. 2B). Thus,
these data suggest that rm-hsc71m expression is
regulated temporally and spatially during early development, such that
early, low expression begins at day 3 in the head and somites and
maximal expression occurs in the entire area of the trunk starting
at day 10. ![Figure 2 Figure 2](picrender.fcgi?artid=55811&blobname=gke43202.gif) | Figure 2 Expression of the rm-hsc71m gene
is modulated during early development. (A) Expression
of rm-hsc71m during early development. Total RNAs
were prepared from developing embryos or larvae at each stage of
development (n = 3–10) and used
(more ...) |
To determine whether rm-hsc71m has any tissue-tropism
in its expression in adult fish, RT–PCR analysis was performed with
RNAs isolated from adult tissues. As shown in Figure 3A, the strongest expression was in skeletal
muscle, whereas lower expression levels were in the gill, eye and
brain. Such tissue-specific expression was confirmed by RNA in
situ hybridization of paraffin-sectioned 1-year-old fish. Similar
to RT–PCR analysis, expression of rm-hsc71m was
prominent in skeletal muscle (Fig. 3B, panels
b, e and i), whereas lower expression levels were seen in gill filament
and brain tissues (Fig. 3B, panels b and
g). Thus, our data indicate that rm-hsc71m expression
is regulated from early embryonic stages to adult stages with strong
tissue-tropism. The data also suggest that rm-hsc71m may
play a role in development of skeletal muscle or in the maintenance
of muscle tissue in adult Rivulus. ![Figure 3 Figure 3](picrender.fcgi?artid=55811&blobname=gke43203.gif) | Figure 3 rm-hsc71m expression
is differentially regulated in a tissue-specific manner. (A)
Expression profile of rm-hsc71m in adult tissues.
Total RNAs were isolated from several organs of a 1-year-old fish
and subjected to RT–PCR analysis using a (more ...) |
Identification of the enhancer region for the muscle-specific
expression of rm-hsc71m To identify specific regions of the genomic clone containing elements
responsible for high muscle-specific expression of rm-hsc71m,
we used transient transfection of Rivulus primary skeletal muscle
tissue. Liver tissue, in which expression of rm-hsc71m is
negligible, was included as a negative control. Tissue culture was
conducted as described in Materials and Methods. Following co-transfection
of varying amounts of pCMV-EGFP with a constant amount of pCMV-lacZ,
EGFP expression was measured by RT–PCR and normalized to
lacZ expression. EGFP expression was always proportional to the concentration
of pCMV-EGFP (data not shown), indicating that despite its low efficiency,
transfection of cultured fish tissues is feasible. To identify skeletal muscle regulatory elements, transfections
were performed using 5′ deletion fragments
containing the rm-hsc71m promoter linked to the
EGFP reporter. The activity of each construct was expressed relative
to that of the pRM79 construct (Fig. 4A). Transcriptional
activity of the pRM79 construct containing an E-box and a TATA box
was similar in both liver and muscle (data not shown). The relative transcriptional
activity of pRM2792 and pRM2654 in muscle tissue was 3-fold higher
than in liver. However, a 5′ deletion
up to –1943 (pRM1943) reduced the transcriptional activity
in muscle to a level seen in liver. Deletion of downstream sequences
(–1572, –1278, –775, –389 and –214)
did not significantly reduce promoter activity in either tissue.
These data indicate that a 712 bp region between –2654
and –1943 functions as a muscle-specific element in the rm-hsc71m gene; therefore we designate this region
as a Rivulus major muscle element (rMME). ![Figure 4 Figure 4](picrender.fcgi?artid=55811&blobname=gke43204.gif) | Figure 4 Identification of a muscle-specific
regulatory region in rm-hsc71m upstream sequences.
(A) Examination of the activity of 5′-upstream
sequences in muscle based on reporter activity. Schematic drawings
of a series of 5′-deletion constructs
(more ...) |
To confirm that rMME is the major muscle-specific regulatory
element, two reporters (pRM1943-3′Sen
and pRM1943-3′Asen) containing rMME
cloned 3′ of the reporter in both sense
and antisense orientations, respectively, were constructed and analyzed
in the transient assay (Fig. 4B). Activation
by pRM1943-3′Sen was comparable to that
of RM2654, whereas activation by pRM1943-3′Asen
was comparable to that of pRM1943. Thus, this result suggests that the
rMME is a muscle-specific upstream promoter element although we
do not exclude the possibility that the rMME is part of an enhancer
(see Discussion). Identification of muscle-specific factor binding
sites in the rMME of rm-hsc71m To identify specific factors that might regulate rm-hsc71m expression
in muscle, we searched mammalian databases for putative transcription
factor binding sites within the genomic clone. We found several
potential binding sites for muscle-specific factors, namely, four
E-box (CANNTG) motifs, one element for transcriptional enhancer
factor 1 and one myocyte enhancer factor 2/serum response
element ( 43– 46), within the rMME region. Of the four
E-boxes, three are located in sub-region B (from –2417
to –2215 nt), whereas the fourth one is in sub-region A
(Fig. 5A). Interestingly, sequences from –2351
to –2267 nt of sub-region B (rm-hsc71mE box) were similar
to the sequence and spacing of two E-boxes found in the MCKE, which
is one of the best-characterized skeletal muscle cell-specific regulatory
elements ( 45– 47) (Fig. 5A).
Thus, we examined whether binding to the rm-hsc71mE box was similar
to that seen in the MCKE by an electrophoretic shift assay using probes
derived from both the MCK enhancer and rm-hsc71mE. Strong MCKE-binding
activity was observed in both muscle and liver extracts, although
a weaker muscle-specific complex was also observed (Fig. 5B). The strong complex formed on the MCKE probe
was competed with excess amounts of the cold MCKE but not with the
rm-hsc71mE (data not shown). In contrast, a muscle-specific complex
with different mobility from that seen in liver was formed on the
rm-hsc71mE probe with muscle extracts and competed by excess cold
rm-hsc71mE competitor (Fig. 5B, lanes 6–8)
but not by the MCKE competitor (data not shown). This muscle-specific
binding activity is the major one observed in region B (Fig. 5C), and it is also observed with two other
DNA fragments A and C (Fig. 5D).
These complexes were competed by rm-hsc71mE and DNA fragment B cold
competitors (Fig. 5B and data not shown).
It is noteworthy that an E-box motif is not found in region C. Thus,
this investigation suggests that sequences responsible for muscle-specific
rm-hsc71mE binding activity are not E-box motifs and that E-box
binding activity may not be sufficient for muscle-specific high
expression of rm-hsc71m in Rivulus. ![Figure 5 Figure 5](picrender.fcgi?artid=55811&blobname=gke43205.gif) | Figure 5 Identification of muscle-specific
binding activity in the rMME of rm-hsc71m. (A)
Schematic representation of the rMME (from –2654 to –1943)
of rm-hsc71m is shown. Three putative muscle-specific
factor-binding sub-elements identified (more ...) |
To identify specific sequences responsible for muscle-specific
binding activity within the rm-hsc71mE, we searched for homologous
regions on both strands within regions A, B and C, and identified
the consensus sequence TGTnACA (Fig. 6D).
To determine whether these sequences were responsible for muscle-specific
binding activity, electrophoretic gel shift assays were performed
using double-stranded probe DNAs containing conserved sequences
from each of these regions. As a negative control, a right half
E-box from the MCKE (MCKE-RH) was also included. Indeed, as shown
in Figure 6A, a muscle-specific complex,
similar to that found in the rm-hsc71mE probe, was formed in every
reaction containing each probe, but not on the MCKE-RH probe. The binding
affinity was of the order of b box > c box > a
box, and was recapitulated in the competitive gel shift analysis
(Fig. 6B). Furthermore, when a double-stranded
mutant oligonucleotide (m box), in which all of the central conserved
sequences of the b box were altered ( TGTG ACA→ ACAG TGT)
was used as a probe, muscle-specific rm-hsc71mE binding activity
disappeared (Fig. 6C). On the other hand,
the bm box with three base substitutions in the central conserved
region of the b box was a poor probe for the muscle-specific factor,
but was a fairly good competitor for the b box (Fig. 6A and B). These observations suggest that the
central TGTnACA sequence is essential for muscle-specific factor
recognition even though sequences flanking the central TGTnACA may
also participate in binding activity. ![Figure 6 Figure 6](picrender.fcgi?artid=55811&blobname=gke43206.gif) | Figure 6 A novel muscle-specific factor
is responsible for recognition of TGTnACA sequences in all three
5′-distal sub-elements of the rMME of rm-hsc71m. (A) Electrophoretic
mobility shift analysis showing binding specificity of a novel muscle-specific
(more ...) |
Identification of a muscle-specific trans-acting
factor recognizing a TGTnACA box A southwestern assay was employed to identify and determine the
size of the putative muscle-specific factor interacting selectively
with the TGTnACA box DNA. Rivulus muscle and liver S150 extracts
(100 µg) were separated on an SDS–polyacrylamide
gel (Fig. 7A), and a southwestern assay
was performed using the double-stranded probe containing either
central TGTnACA sequences (b box) or mutant ones (m box) (see Fig. 6D). A band with an apparent Mr of
32 000 was detected in muscle extracts but not in liver extracts
to the b box probe (Fig. 7B), whereas no
band was detected in both extracts to the m box probe (Fig. 7C). This protein band was efficiently competed
in a reaction containing excess amounts of cold competitors, such
as DNA from boxes a, b and c, but not by MCKE DNA or by the m box
(data not shown). Thus, these data indicate that a muscle-specific
32 kDa protein is abundant in Rivulus muscle and responsible for
the binding to the TGTnACA box. ![Figure 7 Figure 7](picrender.fcgi?artid=55811&blobname=gke43207.gif) | Figure 7 Identification of the TGTnACA
box-binding protein in Mangrove rivulus muscle by southwestern blotting.
S150 extracts from Mangrove rivulus liver and muscle tissues were
subjected to SDS–PAGE and either stained with Coomassie
brilliant blue (more ...) |
|
References 1. Morimoto R.I.,
Tissieres,A. and Georgopoulos,C. (1994) The
Biology of Heat Shock Proteins and Molecular Chaperones. Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 2. Feder M.E. and
Hofmann,G.E. (1999) Heat-shock proteins, molecular chaperones,
and the stress response: evolutionary and ecological physiology. Annu.
Rev. Physiol., 61:, 243–282. [PubMed]. 3. Sorger P.K. (1991)
Heat shock factor and the heat shock response. Cell, 65:, 363–366. [PubMed]. 4. Boorstein W.R.,
Ziegelhoffer,T. and Craig,E.A. (1994) Molecular evolution
of the HSP70 multigene family. J. Mol. Evol., 38:, 1–17. [PubMed]. 5. Loones M.T. and
Morange,M. (1998) Hsp and chaperone distribution during
endochondral bone development in mouse embryo. Cell. Stress Chaperones, 3:, 237–244. [PubMed]. 6. Masuda H.,
Hosokawa,N. and Nagata,K. (1998) Expression and localization
of collagen-binding stress protein Hsp47 in mouse embryo development:
comparison with types I and II collagen. Cell. Stress Chaperones, 3:, 256–264. [PubMed]. 7. Giudice G.,
Sconzo,G. and Roccheri,M.C. (1999) Studies on heat
shock proteins in sea urchin development. Dev. Growth Differ., 41:, 375–380. [PubMed]. 8. Christians E.,
Michel,E. and Renard,J.-P. (1997) Developmental control of
heat shock and chaperone gene expression. Hsp 70 genes and heat shock
factors during preimplantation phase of mouse development. Cell. Mol. Life Sci., 53:, 168–178. [PubMed]. 9. de
la Rosa E.J., Vega-Nunez,E., Morales,A.V.,
Serna,J., Rubio,E. and
de Pablo,F. (1998) Modulation of the chaperone heat
shock cognate 70 by embryonic (pro)insulin correlates with prevention
of apoptosis. Proc. Natl Acad. Sci. USA, 95:, 9950–9955. [PubMed]. 10. Freeman B.C. and
Morimoto,R.I. (1996) The human cytosolic molecular chaperones
hsp90, hsp70 (hsc70) and hdj-1 have distinct roles in recognition of
a non-native protein and protein refolding. EMBO J., 15:, 2969–2979. [PubMed]. 11. Ziemienowicz A.,
Zylicz,M., Floth,C. and Hubscher,U. (1995) Calf thymus
Hsc70 protein protects and reactivates prokaryotic and eukaryotic enzymes. J. Biol. Chem., 270:, 15479–15484. [PubMed]. 12. Dubrovsky E.B.,
Dretzen,G. and Berger,E.M. (1996) The Broad-complex gene
is a tissue-specific modulator of the ecdysone response of the Drosophila hsp23 gene. Mol.
Cell. Biol., 16:, 6542–6552. [PubMed]. 13. Hunt C.R.,
Parsian,A.J., Goswami,P.C. and Kozak,C.A. (1999) Characterization
and expression of the mouse Hsc70 gene. Biochim. Biophys. Acta, 1444:, 315–325. [PubMed]. 14. Tavaria M.,
Gabriele,T., Kola,I. and Anderson,R.L. (1996) A hitchhiker’s guide
to the human Hsp70 family. Cell. Stress Chaperones, 1:, 23–28. [PubMed]. 15. Hahnel A.C.,
Gifford,D.J., Heikkila,J.J. and Schultz,G.A. (1986) Expression
of the major heat shock protein (hsp70) family during early mouse
embryo development. Teratog. Carcinog. Mutagen., 6:, 493–510. [PubMed]. 16. Lang L.,
Miskovic,D., Lo,M. and Heikkila,J.J. (2000) Stress-induced, tissue-specific
enrichment of hsp70 mRNA accumulation in Xenopus laevis embryos. Cell.
Stress Chaperones, 5:, 36–44. [PubMed]. 17. Delelis-Fanien C.,
Penrad-Mobayed,M. and Angelier,N. (1997) Molecular
cloning of a cDNA encoding the amphibian Pleurodeles
waltl 70-kDa heat-shock cognate protein. Biochem. Biophys.
Res. Commun., 238:, 159–164. [PubMed]. 18. Perkins L.A.,
Doctor,J.S., Zhang,K., Stinson,L., Perrimon,N. and Craig,E.A. (1990)
Molecular and developmental characterization of the heat shock cognate
4 gene of Drosophila melanogaster. Mol.
Cell. Biol., 10:, 3232–3238. [PubMed]. 19. Walsh D.,
Li,Z., Wu,Y. and Nagata,K. (1997) Heat shock and the
role of the HSPs during neural plate induction in early mammalian
CNS and brain development. Cell. Mol. Life Sci., 53:, 198–211. [PubMed]. 20. Vega-Nunez E.,
Pena-Melian,A., de la Rosa,E.J. and de Pablo,F. (1999) Dynamic
restricted expression of the chaperone Hsc70 in early chick development. Mech.
Dev., 82:, 199–203. [PubMed]. 21. Zafarullah M.,
Wisniewski,J., Shworak,N.W., Schieman,S., Misra,S. and Gedamu,L.
(1992) Molecular cloning and characterization of a constitutively
expressed heat-shock-cognate hsc71 gene from rainbow trout. Eur. J. Biochem., 204:, 893–900. [PubMed]. 22. Santacruz H.,
Vriz,S. and Angelier,N. (1997) Molecular characterization of
a heat shock cognate cDNA of zebrafish, hsc70,
and developmental expression of the corresponding transcripts. Dev.
Genet., 21:, 223–233. [PubMed]. 23. Eisen J.S. (1996)
Zebrafish make a big splash. Cell, 87:, 969–977. [PubMed]. 24. Nusslein-Volhard C. (1996)
Gradients that organize embryo development. Sci.
Am., 275:, 54–61. [PubMed]. 25. Harrington R.W.,Jr (1961)
Oviparous hermaphroditic fish with self internal fertilization. Science, 134:, 1749–1750. 26. Laughlin T.F.,
Lubinski,B.A., Park,E.-H., Taylor,D.S. and Turner,B.J. (1995)
Clonal stability and mutation in the self-fertilizing hermaphroditic fish, Rivulus marmoratus. J. Hered., 86:, 399–402. [PubMed]. 27. Harrington R.W.,Jr (1971)
How ecological and genetic factors interact to determine when self-fertilizing
hermaphrodites of Rivulus marmoratus change into
functional secondary males, with a reappraisal of the modes of intersexuality
among fishes. Copeia, 1971:, 389–432. 28. Lindsey C.C. and
Harrington,R.W.,Jr (1972) Extreme vertebral variation induced
by temperature in a homozygous clone of the self-fertilizing fish Rivulus marmoratus. Can.
J. Zool., 50:, 733–744. 29. Kim A.-R.,
Kweon,H.-S., Noh,J.-K., Choi,M.-G. and Park,E.-H. (1993) Tolerance
to acidic and alkaline water of laboratory-reared hermaphroditic
fish Rivulus maromoratus. The
4th Indo-Pacific Fish Conference Program and Abstracts of Papers,
p. 114. 30. Lee J.-S.,
Choe,J. and Park,E.-H. (1994) Absence of the intron-D-exon
of c-Ha-ras oncogene in the hermaphroditic fish Rivulus
marmoratus (Teleostei: Rivulidae). Biochem.
Mol. Biol. Int., 34:, 921–926. [PubMed]. 31. Williams J.B. and
Mason,P.J. (1985) Primer extension methods. In Homes,B.D.
and Higgins,S,J. (eds), Nucleic Acid Hybridization:
A Practical Approach. IRL Press, Oxford, UK,
pp. 139–160. 32. Park E.-H. and
Kim,D.S. (1984) Hepatocarcinogenicity of diethylnitrosamine to
the self-fertilizing hermaphroditic fish Rivulus marmoratus (Teleostomi: Cyprinodontidae). J. Natl Cancer Inst., 73:, 871–876. [PubMed]. 33. Park E.-H. and
Yi,A.-K. (1989) Photoreactivation rescue and dark repair demonstrated
in UV-irradiated embryos of the self-fertilizing fish Rivulus marmoratus (Teleostei;
Aplocheilidae). Mutat. Res., 217:, 19–24. [PubMed]. 34. Harrington R.W.,Jr (1963)
Twenty-four hour rhythms of internal self-fertilization and of oviposition
by hermaphrodites of Rivulus marmoratus. Physiol.
Zool., 36:, 325–341. 35. Kim C.G. (1996)
Knock-out effects of transcription factor GATA-1 on early erythropoiesis. Mol.
Cells, 6:, 176–182. 36. Roy R.J.,
Gosselin,P. and Guerin,S.L. (1991) A short protocol
for micro-purification of nuclear proteins from whole animal tissue. Biotechniques, 11:, 770–777. [PubMed]. 37. Kim C.G.,
Swendeman,S.L., Barnhart,K.M. and Sheffery,M. (1990) Promoter
elements and erythroid cell nuclear factors that regulate alpha-globin
gene transcription in vitro. Mol. Cell. Biol., 10:, 5958–5966. [PubMed]. 38. Silva C.M.,
Tully,D.B., Petch,L.A., Jewell,C.M. and Cildowski,J.A. (1987)
Application of a protein-blotting procedure to the study of human glucocorticoid
receptor interactions with DNA. Proc. Natl Acad Sci. USA, 84:, 1744–1748. [PubMed]. 39. Harland R.M. (1991) In situ hybridization: an improved whole-mount method
for Xenopus embryos. Methods Cell Biol., 36:, 685–695. [PubMed]. 40. Stern C.D. and
Holland,P.W. (1993) Essential Developmental
Biology: A Practical Approach. IRL Press, Oxford, UK. 41. Groman D.B. (1982) Histology of the Striped Bass, Monograph
number 3. American Fisheries Society, Bethesda, MD, USA. 42. Springer J.E.,
Robbins,E., Gwag,B.J., Lewis,M.E. and Baldino,F.,Jr (1991)
Non-radioactive detection of nerve growth factor receptor (NGFR) mRNA
in rat brain using in situ hybridization histochemistry. J. Histochem.
Cytochem., 39:, 231–234. [PubMed]43. Perry R.L. and
Rudnicki,M.A. (2000) Molecular mechanisms regulating myogenic
determination and differentiation. Front. Biosci., 5:, D750–D767. [PubMed]. 44. Cossu G. and
Borello,U. (1999) Wnt signaling and the activation
of myogenesis in mammals. EMBO J., 18:, 6867–6872. [PubMed]. 45. Buskin J.N. and
Hauschka,S.D. (1989) Identification of a myocyte nuclear
factor that binds to the muscle-specific enhancer of the mouse muscle
creatine kinase gene. Mol. Cell. Biol., 9:, 2627–2640. [PubMed]. 46. Lassar A.B.,
Buskin,J.N., Lockshon,D., Davis,R.L., Apone,S., Hauschka,S.D. and
Weintraub,H. (1989) MyoD is a sequence-specific DNA
binding protein requiring a region of myc homology
to bind to the muscle creatine kinase enhancer. Cell, 58:, 823–831. [PubMed]. 47. Apone S. and
Hauschka,S.D. (1995) Muscle gene E-box control elements. Evidence
for quantitatively different transcriptional activities and the binding
of distinct regulatory factors. J. Biol. Chem., 270:, 21420–21427. [PubMed]. 48. Naya F.S. and
Olson,E. (1999) MEF2: a transcriptional target for signaling
pathways controlling skeletal muscle growth and differentiation. Curr.
Opin. Cell Biol., 11:, 683–688. [PubMed]. 49. Weintraub H. (1993)
The MyoD family and myogenesis: redundancy, networks, and thresholds. Cell, 75:, 1241–1244. [PubMed]. 50. Mori K.,
Kawahara,T., Yoshida,H., Yanagi,H. and Yura,T. (1996) Signaling
from endoplasmic reticulum to nucleus: transcription factor with
a basic-leucine zipper motif is required for the unfolded protein-response
pathway. Genes Cells, 1:, 803–817. [PubMed]. 51. Cox J.S. and
Walter,P. (1996) A novel mechanism for regulating activity of
a transcription factor that controls the unfolded protein response. Cell, 87:, 391–404. [PubMed]. 52. Nikawa J.,
Akiyoshi,M., Hirata,S. and Fukuda,T. (1996) Saccharomyces cerevisiae IRE2/HAC1
is involved in IRE1-mediated KAR2 expression. Nucleic Acids
Res., 24:, 4222–4226. [PubMed]. 53. Mori K.,
Sant,A., Kohno,K., Normington,K., Gething,M.J. and Sambrook,J.F.
(1992) A 22 bp cis-acting element
is necessary and sufficient for the induction of the yeast KAR2
(BiP) gene by unfolded proteins. EMBO J., 11:, 2583–2593. [PubMed]. 54. Kohno K.,
Normington,K., Sambrook,J., Gething,M.J. and Mori,K. (1993)
The promoter region of the yeast KAR2 (BiP) gene contains a regulatory
domain that responds to the presence of unfolded proteins in the
endoplasmic reticulum. Mol. Cell. Biol., 13:, 877–890. [PubMed]. 55. Mori K.,
Ogawa,N., Kawahara,T., Yanagi,H. and Yura,T. (1998) Palindrome
with spacer of one nucleotide is characteristic of the cis-acting unfolded
protein response element in Saccharomyces cerevisiae. J. Biol. Chem., 273:, 9912–9920. [PubMed]. |