|
FIG. 1. ACHE 5' upstream sequence. DNA sequencing was
performed as detailed (8) on single- and double-stranded DNA by
using commercial (Stratagene) or unique primers determined by
sequencing. Data analysis (N-SITES program, University of Wisconsin)
revealed potential binding sites for several transcription factors,
including MyoD (nt 37,91, 129, 226, 234,348, 1332, and 2130; boxed),
Spl (nt 515, 521, 534, 540, 544, 699, 805, 994, 1246, 1358, 1481, and
1762, circled), and additional sites (underlined): ATF/CREB (nt 67),
E-box (nt 89), Zeste (nt 213 and 855), AP-2 (nt 443 and 1444), GAGA
(nt 477), EGR1 (nt 517, 536, and 546), NF-KB (nt 705 and 1372),
CCAAT box (nt 990 and 1666), and the TATA box (nt 1176). Arrows
indicate potential sites for splicing at the 5' acceptor site (G/GT). The
consensus splicing motif (CAG/A) required at the 3' boundaries of
introns is noted by an arrowhead. One of several putative cap sites
is noted by a [ sign. PL /, polylinker including a Sac I site.
|
|
FIG. 2. Transcription of HpACHE in Xenopus. (Upper) Injected
HpACHE DNAs included the 2.2-kb AcChoEase upstream sequence
or its deletion constructs linked to the AcChoEase coding sequence.
Characteristic restriction sites (S, Sac I; X, Xho I; A, Acc I; K, Kpn
I; P, Pvu II; SP, Sph I; St, Stu I) included in the AcChoEase coding
exons (exons 2, 3, 4, and 6, shaded boxes) and in the region upstream
of the initiator AUG codon within clone GNACHE (8) were predicted
according to sequence data and confirmed by blot hybridization
of genomic DNA with PCR-amplified or subcloned probes from
this sequence. Relative positions are noted on the length scale in kb,
where the first sequenced nucleotide is numbered zero. A fifth
construct, carrying the AcChoEase coding sequence downstream of
the cytomegalovirus immediate early gene enhancer-promoter sequence
(solid box), was as described (17). Putative TATA box and
cluster of transcription factor binding sites (Fig. 1) are marked by a
solid box and an asterisk, respectively. Open reading frames in exons
are noted by dotted underlines. Positions of the first exon (exon 1)
and intron (intron I1) were determined by RNA-PCR experiments
(Fig. 3). (Lower) RNA was extracted from Xenopus oocytes 2 days
after injection (50 ng per oocyte) with the noted linearized constructs.
Residual injected DNA was eliminated by DNase I treatment [20 min
at 37°C, 2 units per sample (Promega)] in 40 mM Tris.HCl/10 mM
NaCl/6 mM MgCl2 in the presence of RNasin (Boehringer Mannheim;
20 units per sample). DNase I was heat-inactivated (90°C, 8
min) and RNA-PCR amplification was performed using the primer
pair 11/12. Lanes: U, uninjected; P, X, K, and S, constructs (named
after the restriction enzymes used to prepare them) (Upper); C,
CMVACHE; M, DNA size marker VI (Boehringer Mannheim). PCR
products are indicated by the arrows.
|
|
FIG. 3. RNA-PCR analysis of exon-intron boundaries. (Upper)
Potential 5' splice sites (consensus G/GT) were found in the AcCho-
Ease upstream region at the noted positions in the sequence presented
in Fig. 1. A potential 3' splice site was known to be located
at position 2226, 21 nt upstream from the first AUG (8). Chimeric
downstream-oriented (+) PCR primers were computer-designed (20)
to function only if their entire consecutive sequence would be
present-i.e., wherever splicing occurred. These included 15-21 nt
upstream from each potential 5' splice site and an additional 5 or 6
nt from exon 2 (ACGCCG, nt 2227-2232) and were tested with a
single upstream (-) primer (primer 10). The asterisk indicates the
experimentally confirmed active 5' splice site. (Lower Right) Total
RNA was extracted from adult human (70 years old) brain basal
nuclei. RNA-PCRs with the chimeric primer 2 and standard PCR
primers 6-8 resulted in PCR products with increasing lengths (lanes
2 and 6-8). An additional primer at position 590 remained inactive
(data not shown), delineating 74 bp for El. Lane M contains
molecular size markers (Boehringer Mannheim). Lanes 1 and 3-5
contain brain RNA and chimeric primers 1 and 3-5, which remained
inactive when paired with primer 10. (Lower Left) RNA extracted
from Xenopus oocytes (lanes XO) and 2-day embryos (lanes XE)
injected with the HpACHE (lanes S) construct (Fig. 2) was used for
RNA-PCR experiments using primers 2 and 10. Lane U, uninjected.
|
|
FIG. 4. AcChoEase expression in developing Xenopus embryos. (A) Schematic presentation of sectioned embryos. Thin sections (700 A)
were prepared from the anterior trunk region (Upper) of 2-day embryos. Cross sections (Lower) at this level reveal the close proximity of the
muscle-forming myotomes and the principal components of the developing central nervous system. At this stage embryos displayed clearly
differentiated muscle cells and the sporadic twitching that accompanies hatching. After cytochemical staining for catalytically active AcChoEase,
electron microscopy was employed to detect high-density accumulations of thiocholine reaction product in myotome areas (shaded). (Diagrams
are after ref. 21.) (Bar = 200 ,um.) (B) Accumulation of AcChoEase in myotomes of transgenic Xenopus embryos. Two-cell cleaving embryos
of Xenopus laevis microinjected with 1 ng of plasmid DNA were cultured for 2 days. Shortly after hatching [stage 26 (21)], embryos were fixed
and stained for catalytically active AcChoEase by the thiocholine hydrolysis method (14, 15). Deposits of electron-dense reaction product appear
as dark rectangular crystals that vary in size and intensity (arrows). Control sections incubated in reaction buffer lacking substrate displayed
no reaction product. Panels a-c show neuromuscular junctions (nmj) demonstrating uncharacteristic synaptic accumulation of AcChoEase after
microinjection of HpACHE (panel b) and CMVACHE (panel c) DNAs. Panels: a, uninjected; b, HpACHE injected; c, CMVACHE injected.
Sections through somitic muscle cells from control uninjected embryos (panel d) or HpACHE-injected (panel e) or CMVACHE-injected (panel
f) embryos illustrating overexpression of AcChoEase around and between the myofibrils (mf) of both DNA-injected groups. Arrows denote
crystals of reaction product. sv, Synaptic vesicles; y, yolk platelets. (Bar = 1 ,um.)
|
