Previous Article | Next Article 
Mol Cell Biol, April 1998, p. 2382-2391, Vol. 18, No. 4
Division of Basic Sciences, Fred Hutchinson
Cancer Research Center, Seattle, Washington 98109-1024
Received 22 August 1997/Returned for modification 16 October
1997/Accepted 2 January 1998
Germ line transformation of white Matrix attachment regions (MARs) are
DNA elements that are identified and defined by their ability to bind
to DNA- and histone-depleted nuclei, which are generally termed nuclear
matrices (9, 33). MARs are typically AT-rich elements that
contain consensus cleavage sites for topoisomerase II, and they may
contain one or more loosely defined short sequence motifs, but, in
general, their structures are not highly homologous. MARs are dispersed
throughout eukaryotic genomes, having been found in centromeric DNA
(47), within genes (9, 10, 20, 22, 40), and in
intergenic regions (4, 11, 14, 20, 29, 33, 35). The
matrix-binding activities of MARs have been conserved throughout
eukaryotic evolution (9, 19). The functions of MARs in vivo
are largely unknown, but one commonly held view is that MARs anchor
individual chromatin loops to a proteinaceous matrix or scaffold in
both interphase nuclei (14, 33, 47) and mitotic chromosomes
(46).
An increasing body of evidence suggests that MARs may play a direct
role in the regulation of gene expression. For example, the intronic
MAR of the immunoglobulin Another putative function of MAR elements, particularly those that
flank individual genes or gene clusters, is to act as insulator elements. This is an attractive hypothesis because it equates the
structural boundaries of a chromatin loop, the flanking MAR elements,
with the functional boundaries of the domain, the putative chromosomal
insulator elements. According to this hypothesis, MAR elements, or
other elements at chromatin domain boundaries, may act as insulators,
shielding genes within the domain from the regulatory elements of
adjacent domains. However, this hypothesis has been difficult to test
experimentally. Studies designed to test the ability of MARs to
insulate transgenes from position effects have been reported in both
plant (1, 6, 43, 51, 52) and animal systems (28, 31,
32, 34, 38, 45, 48, 49). While many of these studies have shown
that transgenes flanked by MARs are more highly expressed than similar
transgenes without MAR elements, conflicting views have been expressed
as to whether MAR elements can render transgene expression
position-independent. For example, different groups have reported that
transgene expression from concatemeric arrays was silenced (1,
21), expressed in a copy number-dependent fashion (5, 15,
31, 36, 37, 45), or neither (38). These conflicting
views are due, at least in part, to the inherent limitations of these
transfection assays, because the numbers and arrangements of transgene
sequences within the typically multimeric arrays are difficult to
determine and could differ in a number of ways. First, the number and
arrangement of transgenes within a single concatemeric array could
affect transgene silencing versus activation. Second, some of the
transfectant clones that have been studied contained multiple transgene
insertions, with different transgene arrays integrated at different
chromosomal sites. Patterns of gene expression among such genotypically
complex transfectants might be difficult to discern. Finally,
rearrangement of transgene sequences was a common event in some
experiments, although transgene expression could still be detected
(1). Therefore, meaningful genotype-phenotype correlations
in such transfectant clones would be difficult to establish.
One reasonable, if inefficient, means to circumvent the limitations
inherent in analyses of transfectants containing multiple transgene
insertions would be to study only those transfectant clones that
contain single, intact transgenes integrated in the recipient cell
genome. We used this approach previously to study the functions of MAR
elements from the human apolipoprotein B-100 (apoB) locus
(21). The human apoB gene is thought to be the sole resident
of a 48-kb DNase I-sensitive domain that is flanked by MAR elements
(29). When single-copy transfectants containing lacZ reporters with or without flanking apoB MARs were
analyzed, a significant increase in transgene expression and a
reduction in variability of expression among apoB MAR-containing clones were observed (21). This was consistent with the suggestion that the apoB MARs were insulating transgene expression from
chromosomal position effects, although the possibility that the MARs
resulted in integration-dependent enhancement could not be excluded by these data.
To more critically test the possibility that the apoB MARs were
functional boundaries of the apoB domain, we studied their insulating
activities in a position effect assay in Drosophila melanogaster, as first described by Kellum and Schedl
(23). In this assay, germ line transformation of
white Construction of transformation vectors.
The
P-element transformation vector RW+, herein
designated EmwS', was kindly provided by Paul Schedl; the construction
of this plasmid has been described (53). apoB3'MEmwS' was
generated by ligating a 786-bp XhoI fragment containing the
apoB 3' MAR (29) into the unique XhoI site
upstream of white in EmwS'. The orientation of the apoB 3'
MAR fragment in the various subclones was determined by restriction
analysis. The P-element vector with scs upstream
of white (SEmwS') was made by directional ligation of a
gel-purified 1.7-kb KpnI/SalI fragment containing
scs (23, 50) into the unique
KpnI/XhoI site upstream of white in
EmwS'. SmwS' was generated by digesting EmwS' with KpnI and
SpeI, which removed the white upstream regulatory
region, and inserting a gel-purified 1.7-kb
KpnI/SpeI fragment containing scs into
the linearized plasmid at this site. This resulted in the replacement of the white upstream regulatory region with scs.
All of the other constructs in which the white
upstream regulatory region was deleted were derivatives of Transformation and line establishment.
Samples (500 µg/ml)
of each P-element transformation vector were coinjected with
150 µg of helper plasmid p Eye pigment assay.
Five mating pairs of flies for each line
were placed at 25°C. After 5 days the adults were removed to prevent
overcrowding of larvae, which can result in variations in head and body
size among the progeny. Heterozygous virgin females were then collected and aged for 6 days at 25°C. To quantitate eye pigment, flies from
each phenotypic class were collected, frozen, and decapitated by
vortexing for 10 s. Heads from each eye color category (13 for
pale yellow [phenotypic class I], 10 for yellow [II], 8 for orange
[III], 5 for dark orange [IV], and 4 for red [V] and dark red
[VI]) were pooled, and pigment was extracted by incubating the heads
in 30% ethyl alcohol, pH 2, at room temperature for 4 days. Pigment
absorption was determined at 450 nm. Each group of heads was assayed in
triplicate, and absorption-per-head values were determined.
Photography.
The eyes of 4-day-old heterozygous females were
photographed using a Zeiss SR microscope fitted with a Nikon FX-35 WA
camera. Illumination was supplied by a Nikon MK II fiber optic light
source, and photographic images were prepared with Fujichrome tungsten 64T film. Transformed lines carrying P-element insertions in
the X chromosome were backcrossed to w1118
stocks to remove the In(1)FM6,B balancer chromosome prior to photography. The eye color phenotypes were identical in both genetic backgrounds.
The human apoB 3' MAR functions as an insulator element in
Drosophila.
The P-element transformation vectors
used in this study are shown in Fig. 1.
In the first set of experiments, three different P-element
transposons were employed. EmwS' contained the white gene
promoter and enhancer (E), a white cDNA coding cassette
(termed mini-white [mw]), and the Drosophila
scs' (S') element downstream of the white transcription
unit (Fig. 1a). In addition, SEmwS' (Fig. 1b) contained the
Drosophila scs element (S) upstream of white, and
apoB3'MEmwS' (Fig. 1c) contained the 3' MAR from the human apoB
locus (apoB3'M) inserted upstream of white.
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Human Matrix Attachment Regions Insulate Transgene
Expression from Chromosomal Position Effects in Drosophila
melanogaster
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
Drosophila embryos with P-element vectors containing
white expression cassettes results in flies with different
eye color phenotypes due to position effects at the sites of transgene
insertion. These position effects can be cured by specific DNA
elements, such as the Drosophila scs and scs'
elements, that have insulator activity in vivo. We have used this
system to determine whether human matrix attachment regions (MARs) can
function as insulator elements in vivo. Two different human MARs, from
the apolipoprotein B and 
1-antitrypsin loci, insulated
white transgene expression from position effects in
Drosophila melanogaster. Both elements reduced variability in transgene expression without enhancing levels of white
gene expression. In contrast, expression of white
transgenes containing human DNA segments without matrix-binding
activity was highly variable in Drosophila transformants.
These data indicate that human MARs can function as insulator elements
in vivo.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
gene is adjacent to a tissue-specific
enhancer, and both elements are required for the proper regulation of
the immunoglobulin gene during development (24, 30).
Deletion of either the MAR or the enhancer resulted in constitutive
hypermethylation of the gene in all cell types and in permanent
repression of the locus (24). Moreover, replacement of the
intronic MAR with MARs from other locations in the genome or from
other species restored the normal pattern of both methylation and gene
expression. These results indicate that MARs can be directly involved
in the regulation of gene expression, and they also suggest that MAR
function may be neither tissue nor species specific.
(w)
Drosophila embryos with P-element vectors
containing white transgenes results in transgenic flies with
different eye color phenotypes, as each white transgene is
expressed at levels dictated by regulatory elements at the site of
insertion (17, 27). In contrast, P elements
containing white transgenes that are flanked by
insulating elements, such as the specialized chromatin structures
(scs and scs') from the hsp70 locus of
Drosophila (50), are expressed in a
position-independent manner, and all of the transgenic lines display
similar eye color phenotypes (23). A vertebrate regulatory element, hypersensitive site 4 (HS4) of the chicken 
-globin locus control region (LCR), also functions as an insulator in this assay (8). In this study, we used the Drosophila assay
to assess the insulating properties of human MAR elements. MARs from
the human apoB and 1-antitrypsin (1AT) loci displayed
insulating activities much like those of scs itself. In
contrast, human DNA segments without matrix-binding activity had no
insulating activity in this assay. These results indicate that at least
some human MAR elements can function as chromosomal insulators in vivo.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
XS,
herein designated mwS', which was provided by Paul Schedl. mwS' was
prepared from EmwS' by removing an XhoI/SpeI fragment from the white upstream regulatory region.
apoB3'MmwS', ATRMmwS', apoBmwS', and apoB5'MmwS' were generated
by blunt-end ligation of the appropriate restriction fragments into the
unique XbaI site upstream of white in mwS'. The
inserts used in these constructions were a 786-bp XhoI
fragment containing the apoB 3' MAR (29) for apoB3'MmwS'; a
4.1-kb XhoI/SalI fragment containing the ATR MAR
(39a) for ATRMmwS'; an 800-bp
EcoRI/NcoI fragment containing part of
intron 5, exon 6, and part of intron 6 of the apoB gene (3)
for apoBmwS'; and a 1.0-kb XbaI/XhoI fragment containing the apoB 5' MAR (29) for apoB5'MmwS'. The
orientations of the inserted DNA fragments were determined by
restriction analysis. The constructs without the scs'
element, apoB3'Mmw and apoB3'MEmw, were prepared by deleting an
~400-bp PvuII/SalI fragment from mwS'.
25.7wc2-3 per ml into
w1118 embryos as described by Spradling and
Rubin (44). Survivors were crossed to each other in groups
of five (three females and two males), and transformants were
identified by eye pigmentation. Mixed populations of transgenic flies
were separated on the basis of eye color. Chromosome assignments of the
various transgene insertions were made by crossing the transformants
with w balancer stocks containing dominant
markers: In(2LR)O,Cy for the second chromosome,
In(3LR)TM3,Sb for the third chromosome, and
In(1)FM6,B for the X chromosome. All lines were maintained as balanced stocks. Transgene copy number was determined by Southern hybridization. Genomic DNA was isolated from 20 flies of each transformed line, digested with a restriction endonuclease that cuts
once within the transgene (XbaI for white
enhancer-containing constructs and for mwS'; SpeI for all
other constructs), separated on 1% agarose gels by field
inversion gel electrophoresis, and transferred to nylon membranes in
0.2 M NaOH-0.6 M NaCl denaturing solution. The filters were hybridized
with various radiolabeled DNA probes: a 786-bp XhoI fragment
containing the apoB 3' MAR for apoB3'MEmwS' and apoB3'MmwS', a 4.1-kb
XhoI/SalI fragment containing the ATR MAR for
ATRMmwS', a 1.0-kb XbaI/XhoI fragment containing
the apoB 5' MAR for apoB5'MmwS', and a 400-bp
EcoRI/BamHI fragment containing scs'
for EmwS', mwS', SEmwS', and SmwS'.
RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
View larger version (17K):
[in a new window]
FIG. 1.
white P-element transformation vectors.
Symbols: E, the upstream regulatory region of white that
contains the eye- and testes-specific enhancers; mw,
mini-white cDNA expression cassette which includes the
proximal promoter, with the white transcription start site
depicted by the arrow; S', scs'; S, scs; 3', apoB
3' MAR; 5', apoB 5' MAR; ATR, ATR MAR; apo, apoB transcribed sequence;
black rectangles at the ends of each transposon, 5' and 3' P
elements. Vectors: a, EmwS'; b, SEmwS'; c, apoB3'MEmwS'; d, mwS'; e,
SmwS'; f, apoB3'mwS'; g, ATRMmwS'; h, apoB5'MmwS'; i, apoBmwS'; j,
apoB3'MEmw; and k, apoB3'Mmw.
Drosophila embryos, and
transformed lines were established from flies with any detectable eye
pigment. Transgene copy number was determined in each transformed
line by Southern hybridization (Fig. 2),
and the eye color phenotypes of age-matched females heterozygous for the various white insertions were compared. Each
transformant was assigned to one of six phenotypic classes: I (light
yellow), II (yellow), III (light orange), IV (orange), V (red), and VI (dark red), as shown in Fig. 3,
4, and 5.
Seven transformed lines were obtained using the control construct
EmwS', and all seven lines contained single transgene insertions (data
not shown). As shown in Fig. 3a, three of these lines had light orange
eyes (phenotypic class III), three had red eyes (V), and one had dark red eyes (VI). Thus, transgenes without an insulating element upstream
of white were expressed at different levels in different transformed lines. This observation suggested that the white
transgenes in these lines were sensitive to position effects at the
sites of insertion. These results are in accord with those of Kellum and Schedl (23), who showed that transformants containing
white transgenes without insulating elements had eye color
phenotypes that ranged from orange to dark red. In contrast, all three
single-copy lines that contained SEmwS', in which the white
transcription unit is flanked by scs and scs',
had dark red (VI) eyes (Fig. 3b). This suggested that expression of
white transgenes containing scs and
scs' was largely position independent, as shown previously by Kellum and Schedl (23).
|
|
|
|
The human apoB 3' MAR insulates but does not enhance white gene expression. To distinguish between the possibilities that the human apoB 3' MAR was acting as an insulator versus an enhancer, we prepared P-element transformation vectors in which putative enhancement of white gene expression by the apoB MAR would be readily apparent. To do this, constructs similar to those employed in the experiments described above but lacking the white gene enhancers were prepared. The vectors mwS', SmwS', and apoB3'MmwS' (Fig. 1d, e, and f) were similar to EmwS', SEmwS', and apoB3'MEmwS' (Fig. 1a, b, and c), respectively, but they lacked an ~1.6-kb XhoI/SpeI DNA fragment upstream of mini-white that contains the white gene enhancers (53). white gene expression from such P-element vectors is reduced compared to that from enhancer-containing vectors, so that enhancement of white gene expression can be readily distinguished from insulation (23).
Nineteen mwS' transformants were isolated, and the genotypes of the transformed lines were determined by Southern hybridization. DNA from each transformant was digested with XbaI, which cuts once within the P-element vector, resolved by electrophoresis, and hybridized with a Drosophila scs' probe. Figure 2a shows results for six of the mwS' transformants. As shown in the figure, each line contained an ~10-kb fragment that corresponded to the endogenous scs' element at hsp70. In addition, each transformant contained one (Fig. 2a, lanes 1 to 4) or two (lanes 5 to 6) additional scs' fragments, indicating single or double transgene insertions. In total, 16 of the 19 mwS' transformants contained single P-element insertions. As expected, white gene expression in the mwS' lines, as judged by eye pigmentation, was generally less than that of transformants expressing EmwS', which includes the white enhancers (compare Fig. 3a and 4a). Nonetheless, the eye color phenotypes of the mwS' transformants varied considerably, ranging from light yellow (I) to red (V) (Fig. 4a). The 16 mwS' transformants were widely distributed in five phenotypic classes (I, 1; II, 5; III, 6; IV, 2; and V, 2). Thus, mwS' transposons, like EmwS' vectors, were sensitive to chromosomal position effects when integrated in the Drosophila genome. In contrast, all five lines derived from SmwS'-injected embryos fell into a single phenotypic class, light orange (III) (Fig. 4b), indicating that white expression from this P-element vector, which contains scs and scs', was position-independent. Twenty-four of 27 lines transformed with apoB3'MmwS' (Fig. 1f) contained single P-element insertions (data not shown). Twenty of these 24 lines had yellow (II) or light orange (III) eyes (Fig. 4c). These results were similar to those obtained with apoB3'MEmwS', in that >80% of the transformed lines in each experiment fell into two similar phenotypic classes. Furthermore, only 3 of the 24 apoB3'MmwS' transformants had orange (IV) eyes, and none of them had red (V) eyes, so the apoB 3' MAR was clearly not acting as an enhancer in this system (Fig. 4a versus 4c). Thus, inserting the apoB 3' MAR upstream of the white transcription unit of mwS' enriched for yellow-light orange transformants and eliminated red transformants without enhancing white gene expression. These data indicate that the apoB 3' MAR can function as an insulator element in Drosophila. However, the insulating activity of the apoB MAR appeared to be less than that of scs itself, because variation in white gene expression was reduced but not eliminated.The human ATR MAR functions as an insulator element in
Drosophila.
To determine whether other human MARs can
function as insulator elements in Drosophila, we utilized a
MAR that is located approximately 2 kb downstream of the
1-antitrypsin-related (ATR) sequence on human chromosome 14q32.1.
This MAR is one of five matrix-binding elements that we have identified
in an ~120-kb segment of 14q32.1 that includes three related serine
protease inhibitor genes, 1AT, ATR, and corticosteroid-binding
globulin (CBG) (39; unpublished data). A
P-element vector in which the ATR MAR was inserted upstream
of the white transcription unit (ATRMmwS' [Fig. 1g]) was
prepared and used to transform w
Drosophila embryos. Nine of 10 transformed lines contained
single-copy ATRMmwS' insertions (data not shown). Seven of the nine
single-copy lines had yellow (II) eyes, and two had light orange (III)
eyes (Fig. 5a). This distribution of eye color phenotypes was similar to that observed in apoB3'MmwS' transformants (Fig. 4c), suggesting that the human ATR MAR has insulating properties similar to those of
the apoB 3' MAR.
A putative apoB 5' MAR does not function as an insulator element in Drosophila. MARs have been mapped both upstream and downstream of the human apoB gene, and these elements have been proposed to define the limits of the apoB chromatin domain (29). In view of our finding that the apoB 3' MAR acted as an insulator element in Drosophila, it might have been expected that the apoB 5' MAR would have similar properties. To test this possibility, an ~1-kb DNA fragment reported to contain the 5' apoB MAR was inserted upstream of the white transcription unit in the white enhancerless P-element vector (Fig. 1h). Twelve apoB5'MmwS' transformants were obtained; eight of these contained single transgene insertions and four contained double transgene insertions (Fig. 2b). The eye color phenotypes of the eight single-copy transformants varied considerably, ranging from light yellow (I) to red (V) (Fig. 5b). This range of phenotypes, without enrichment for any particular phenotypic class, was much like that seen in the mwS' transformants (Fig. 4a), which do not contain an insulator element upstream of white. These results indicate that the putative apoB 5' MAR does not function as an insulator element in Drosophila. This observation prompted us to reassess the matrix-binding activity of this human DNA fragment. Despite repeated attempts, we have been unable to detect matrix-binding activity of the apoB 5' MAR DNA fragment in any of the standard assays (9, 33). Furthermore, DNA sequencing studies demonstrated that the "apoB 5' MAR" fragment is not particularly AT-rich, nor does it contain the characteristic features of MAR elements (39a). Therefore, the status of this DNA element as a matrix-associated region is uncertain at present.
A DNA segment from within the apoB gene does not insulate in Drosophila. The results described above suggest that at least some human MARs can function as insulator elements in Drosophila. In contrast, a DNA fragment without matrix-binding activity failed to insulate white gene expression from position effects in Drosophila. To explore this difference further, we prepared a transformation vector in which an ~800-bp human DNA fragment from within the apoB gene was inserted upstream of the white transcription unit (apoBmwS' [Fig. 1i]). This human DNA fragment is clearly devoid of matrix-binding activity. Eight of 11 transgenic lines containing apoBmwS' had single-copy transgene insertions. The eye color phenotypes of these eight transformed lines varied widely, ranging from pale yellow (I) to red (V) (Fig. 5c). These results were similar to those obtained with the other position-sensitive P elements, mwS' (Fig. 3a) and apoB5'MmwS' (Fig. 5b). Therefore, this DNA fragment, which contains intron and exon sequences from the human apoB gene, does not function as an insulator element in Drosophila. Thus, human DNA does not have intrinsic insulating activity in this assay, nor is the insulating phenotype due to a distance effect between the white transcription units and Drosophila genomic elements upstream of the P-element insertions.
scs' affects insulator function of the apoB 3' MAR in Drosophila. It has been reported that the scs' fragment used in these and other mini-white transformation vectors is not fully functional as an insulator element in Drosophila (16, 53). To determine whether scs' was required for insulator function of the apoB 3' MAR to be apparent in our assays, transformation vectors containing the apoB 3' MAR upstream of the white transcription unit, but without scs' downstream, were prepared. Two different transformation vectors were prepared, one containing the white gene enhancers (apoB3'MEmw [Fig. 1j]) and one without the white enhancers (apoB3'Mmw [Fig. 1k]). Four of seven apoB3'M Emw transformants had single P-element insertions (data not shown), and all four of these lines had dark red (VI) eyes (Fig. 3d). This was the same phenotypic class as most of the apoB3'MEmwS' transformants, which contained scs' (Fig. 3c). However, the distribution of eye color phenotypes among the apoB3'Mmw transformants was more complex. Eight of 13 single-copy apoB3'Mmw transformants had yellow (II) eyes (Fig. 4d), like most of the scs'-containing apoB3'MmwS' transformants (Fig. 4c). However, three of the apoB3'Mmw transformants had red (V) eyes, a phenotype that was not observed among 24 apoB3'MmwS' transformants. These observations suggest that white transformation vectors containing the apoB 3' MAR alone are more sensitive to chromosomal position effects than vectors containing both the apoB 3' MAR and scs'.
Eye color phenotypes versus eye pigment expression
quantitative
aspects.
The eye color phenotypes of our transformants varied in a
continuous fashion from light yellow to dark red (Fig. 3, 4, and 5). We
divided these phenotypes into six classes, I through VI, which could be
described as various shades of yellow, orange, and red. To determine
how the different phenotypic classes corresponded to the amount of
pigment in the Drosophila eyes, eye pigment was quantitated
spectrophotometrically in the different phenotypic classes.
Transformants from each phenotypic class were obtained, fly heads were
pooled in triplicate, pigment was extracted, and absorbance at 480 nm
was determined. The mean absorbance per head for each phenotypic class
is shown in Fig. 6. The most common phenotypic classes among the enhancerless white
transformants, light yellow (I), yellow (II), and light orange (III),
differed from each other only about twofold, and the amount of pigment per head varied linearly in this range. Thus, small changes in eye
pigment expression among the enhancerless transformants yielded readily
discriminated eye color phenotypes. The orange (IV), red (V), and dark
red (VI) phenotypes corresponded to approximately 2-, 9-, and 11-fold
more pigment than light orange (III), so that eye color readout was a
less sensitive indicator of white expression in this range.
These data demonstrate that eye pigment expression in our transformants
varied over an approximately 20-fold range, and small variations within
this range could be readily resolved into the different phenotypic
classes. Thus, the eye color phenotypes of white
transformants are very sensitive indicators of white gene
expression, which makes this system particularly useful as a position
effect assay. Among the white enhancerless transformants, the apoB 3' MAR and the ATR MAR both enriched for yellow (II) to light
orange (III) transformants (Fig. 4c and 5a). These phenotypes differed
only about 1.2-fold in eye pigment expression. These observations
provide further support for the conclusion that these two human MARs
can function as insulator elements in Drosophila.
|
| |
DISCUSSION |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
MARs are DNA segments that are defined by their abilities to bind to isolated nuclear matrices in vitro (9, 33). These binding properties are consistent with the view that at least some MAR elements might represent structural boundaries of individual chromatin domains, serving to tether the ends of individual chromatin loops to a proteinaceous nuclear matrix in vivo. One functional activity that is often ascribed to DNA elements that are located at or near chromatin domain boundaries is insulation, an activity by which a DNA sequence prevents interactions between neighboring regulatory elements. This suggests that specific DNA segments between neighboring chromatin domains might have both matrix-binding activity and insulator function. Although this model is consistent with currently available data, it has not been critically tested, and alternate interpretations of the data remain viable (26, 54).
The experiments reported here were designed to test whether human MAR
elements can function as chromosomal insulators in vivo. To do this, we
used the mini-white position effect assay of Kellum and
Schedl (23) to study insulator function of human MAR
elements in Drosophila. This assay was used previously to
demonstrate the insulating properties of scs and
scs', which are well-characterized insulator elements from
Drosophila (23). Using this approach, we have
demonstrated that two different human MARs can insulate transgenes from
position effects in Drosophila. w flies
transformed with P elements in which either the apoB 3' MAR
or the ATR MAR was inserted upstream of a white
transcription unit had substantially less variability in
white transgene expression than control transformants
without human MARs. Furthermore, variability of transgene expression
was reduced without increasing the levels of white transgene
expression. This contrasts with conclusions drawn from transfection
experiments, in which MAR elements have been suggested to increase
transgene expression without eliminating variation in expression levels
(1, 38). However, the interpretation of transfection studies
is complicated by the complex rearrangements of transgene sequences
that generally occur in transfected cells. Our experiments clearly show
that neither the apoB 3' MAR nor the ATR MAR enhanced white
gene expression in Drosophila, as most transformants that
expressed white from enhancerless P-element vectors had yellow or orange eyes irrespective of the presence or
absence of MAR elements. This conclusion is in accord with previous
studies, in which the apoB 3' MAR did not enhance expression of
transiently transfected reporter genes in mammalian cells
(21). Thus, the apoB 3' MAR and the ATR MAR do not contain
associated enhancers, unlike some Drosophila MARs
(14), the chicken lysozyme 5' MAR (45), and MARs
from the human immunoglobulin and µ (10, 30) and beta
interferon (25) genes.
In contrast to results obtained with the apoB 3' MAR and the ATR MAR, insulator activity was not observed in human DNA segments that were devoid of matrix-binding activity. For example, an ~800-bp fragment of the apoB gene that included parts of introns 5 and 6 and all of exon 6 had no insulator function in Drosophila. Furthermore, an ~1,000-bp DNA fragment from the upstream region of apoB also failed to function as an insulator. This result was surprising in view of the fact that this DNA fragment had been reported to have matrix-binding activity; indeed, this element is thought to be the upstream boundary of a 48-kb chromatin domain that contains the apoB gene as its sole resident (29). However, we were unable to detect matrix-binding activity of this DNA fragment in any of the standard assays (9, 33). Moreover, the DNA sequence of this fragment has none of the features that commonly occur in MAR elements, except for a single core binding site for topoisomerase II (42). Thus, we conclude that the putative apoB 5' MAR has neither matrix-binding nor insulator activities, and we view its identification as the upstream boundary of the apoB domain with skepticism. This suggests that the chromatin domain structure of the human apoB locus may be more complex and extensive than previously envisioned.
Both the apoB 3' MAR and the ATR MAR insulated white gene
expression from position effects in Drosophila.
Transformants expressing control vectors without insulator elements
upstream of white generally displayed a wide range of eye
color phenotypes with no apparent enrichment for any particular
phenotypic class. In contrast, most of the transformed lines containing
either human MAR upstream of white fell into a single
phenotypic class, and variation in white gene expression was
greatly reduced. However, a few lines in each collection displayed
slightly different eye color phenotypes. Thus, the human apoB 3' MAR
and the ATR MAR reduced, but did not completely eliminate, variability
in transgene expression. This suggests that, in some lines, insulation
was not complete. This could be due to any of several factors. First,
it has been shown that two Drosophila insulators,
scs and suppressor-of-hairy-wing [su(HW)], can
block the effects of some, but not all, enhancers and repressors in
enhancer-blocking assays (7, 41). These assays are thought
to mimic some aspects of insulator function. Thus, some of our
P-element insertions may have occurred in the vicinity of
strong enhancers or repressors, resulting in incomplete insulation. The
notion that insulator elements may vary in intrinsic activity is also
consistent with the observation that two copies of HS4 of the chicken
-globin locus control region were required for insulator function in
Drosophila (8). Second, recent reports suggest
that scs' is a weaker insulator element than either
scs or su(HW) (16, 53). The
scs' fragment used in those experiments, and in those
described here, is a derivative of a larger scs' fragment
originally employed by Kellum and Schedl (23). This derivative seems to have less insulator activity than the larger scs' fragment, which may account for some of the variation
in white expression in our transformants.
The demonstration that human MARs can function as insulator elements in Drosophila suggests that this activity is evolutionarily conserved. It has already been shown that the matrix-binding activities of MARs from different species are highly conserved (9, 19). However, it is not clear at present whether matrix attachment is required for insulator function. We presume that the two activities are distinct because other insulator elements, including scs, scs', su(HW), and HS4, are not known to have matrix-binding activity. Conversely, not all MAR elements establish boundaries between chromatin domains, as some of them map within expressed genes (9, 10, 20, 22, 40), so it seems unlikely that all MARs will function as insulators. Thus, we anticipate that insulator function and matrix-binding activities will prove to be separable activities, although they may colocalize to discrete DNA fragments in some instances. These issues will be interesting to explore.
Finally, the genomic locations of the two human MAR elements that have
insulator activity are consistent with the possibility that they might
represent the boundaries of individual chromatin domains. The apoB 3'
MAR is just downstream of the apoB transcription unit (29),
but the chromatin configuration of sequences further downstream has not
yet been explored. The ATR MAR is located ~2 kb downstream of ATR, an
antitrypsin-related sequence that may (18) or may not
(2) be a pseudogene. The 1AT gene is located ~32 kb
upstream of the ATR MAR, and the CBG gene is ~33 kb downstream (39). 1AT and CBG are highly expressed in hepatic cells
but differentially expressed in macrophages and intestinal epithelium. The entire region is inactive in most other cell types. This provides an opportunity to determine whether the ATR MAR functions as an insulator between putative 1AT and CBG chromatin domains by
constructing and analyzing specifically modified human chromosomes
using recombination-proficient cell hybrids (12, 13).
| |
ACKNOWLEDGMENTS |
|---|
We thank Julio Vazquez and Paul Schedl for the transformation
vectors pRW+ and pXS. Steve Henikoff, Bob Levis,
Georgette Sass, and Julio Vazquez contributed many helpful discussions.
We thank Ed Giniger, Steve Henikoff, Pierre Rollini, and Steve Tapscott
for their critical reviews of the manuscript and Cathy Ludlow for
excellent technical assistance.
S.J.N. was the recipient of a National Research Service Award (DK09188) from the NIH. These studies were supported by grant GM26449 from the National Institute of General Medical Sciences.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Division of Basic Sciences, A2-025, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave. N., P.O. Box 19024, Seattle, WA 98109-1024. Phone: (206) 667-5217. Fax: (206) 667-6522. email: kfournie{at}fhcrc.org.
| |
REFERENCES |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
| 1. | Allen, G. C., G. Hall, Jr., S. Michalowski, W. Newman, S. Spiker, A. K. Weissinger, and W. F. Thompson. 1996. High-level transgene expression in plant cells: effects of a strong scaffold attachment region from tobacco. Plant Cell 8:899-913[Abstract]. |
| 2. |
Bao, J. J.,
L. Reed-Fourquet,
R. N. Sifers,
V. J. Kidd, and S. L. Woo.
1988.
Molecular structure and sequence homology of a gene related to 1-antitrypsin in the human genome.
Genomics
2:165-173[Medline].
|
| 3. |
Blackhart, B. D.,
E. M. Ludwig,
V. R. Pierotti,
L. Caiati,
M. A. Onasch,
S. C. Wallis,
L. Powell,
R. Pease,
T. J. Knott,
M. L. Chu, et al.
1986.
Structure of the human apolipoprotein B gene.
J. Biol. Chem.
261:15364-15367 |
| 4. |
Bode, J., and K. Maass.
1988.
Chromatin domain surrounding the human interferon- gene as defined by scaffold-attached regions.
Biochemistry
27:4706-4711[Medline].
|
| 5. | Bonifer, C., M. Vidal, F. Grosveld, and A. E. Sippel. 1990. Tissue-specific and position-independent expression of the complete gene domain for chicken lysozyme in transgenic mice. EMBO J. 9:2843-2848[Medline]. |
| 6. |
Breyne, P.,
M. van Montagu,
N. Depicker, and G. Gheysen.
1992.
Characterization of a plant scaffold attachment region in a DNA fragment that normalizes transgene expression in tobacco.
Plant Cell
4:463-471 |
| 7. | Cai, H., and M. Levine. 1995. Modulation of enhancer-promoter interactions by insulators in the Drosophila embryo. Nature 376:533-536[Medline]. |
| 8. |
Chung, J. H.,
M. Whiteley, and G. Felsenfeld.
1993.
A 5' element of the chicken -globin domain serves as an insulator in human erythroid cells and protects against position effect in Drosophila.
Cell
74:505-514[Medline].
|
| 9. |
Cockerill, P. N., and W. T. Garrard.
1986.
Chromosomal loop anchorage of the immunoglobulin gene occurs next to the enhancer in a region containing topoisomerase II sites.
Cell
44:273-282[Medline].
|
| 10. |
Cockerill, P. N.,
M. H. Yuen, and W. T. Garrard.
1987.
The enhancer of the immunoglobulin heavy chain locus is flanked by presumptive chromosomal loop anchorage elements.
J. Biol. Chem.
262:5394-5397 |
| 11. |
Conkling, M. A.,
C.-L. Cheng,
Y. T. Yamamoto, and H. M. Goodman.
1990.
Isolation of transcriptionally regulated root-specific genes from tobacco.
Plant Physiol.
93:1203-1211 |
| 12. | Dieken, E. S., E. M. Epner, S. Fiering, R. E. K. Fournier, and M. Groudine. 1996. Efficient modification of human chromosomal alleles using recombination-proficient chicken/human microcell hybrids. Nat. Genet. 12:174-182[Medline]. |
| 13. | Dieken, E. S., and R. E. K. Fournier. 1996. Homologous modification of human chromosomal genes in chicken B-cell x human microcell hybrids. Methods 9:56-63. [Medline] |
| 14. | Gasser, S. M., and U. K. Laemmli. 1986. Cohabitation of scaffold binding regions with upstream/enhancer elements of three developmentally regulated genes of D. melanogaster. Cell 46:521-530[Medline]. |
| 15. |
Grosveld, F.,
G. B. van Assendelft,
D. R. Greaves, and G. Kollias.
1987.
Position-independent, high-level expression of the human -globin gene in transgenic mice.
Cell
51:975-985[Medline].
|
| 16. |
Hagstrom, K.,
M. Muller, and P. Schedl.
1996.
Fab-7 functions as a chromatin domain boundary to ensure proper segment specification by the Drosophila bithorax complex.
Genes Dev.
10:3202-3215 |
| 17. | Hazelrigg, T., R. Levis, and G. M. Rubin. 1984. Transformation of white locus DNA in Drosophila: dosage compensation, zeste interaction, and position effects. Cell 36:469-481[Medline]. |
| 18. |
Hofker, M. H.,
M. Nelen,
E. C. Klasen,
T. Nukiwa,
D. Curiel,
R. G. Crystal, and R. R. Frants.
1988.
Cloning and characterization of an 1-antitrypsin-like gene 12 kb downstream of the genuine 1-antitrypsin gene.
Biochem. Biophys. Res. Commun.
155:634-642[Medline].
|
| 19. | Izaurralde, E., J. Mirkovitch, and U. K. Laemmli. 1988. Interaction of DNA with nuclear scaffolds in vitro. J. Mol. Biol. 200:111-125[Medline]. |
| 20. | Jarman, A. P., and D. R. Higgs. 1988. Nuclear scaffold attachment sites in the human globin gene complexes. EMBO J. 7:3337-3344[Medline]. |
| 21. | Kalos, M., and R. E. K. Fournier. 1995. Position-independent transgene expression mediated by boundary elements from the apolipoprotein B chromatin domain. Mol. Cell. Biol. 15:198-207[Abstract]. |
| 22. | Kas, E., and L. A. Chasin. 1987. Anchorage of the Chinese hamster dihydrofolate reductase gene to the nuclear scaffold occurs in an intragenic region. J. Mol. Biol. 198:677-692[Medline]. |
| 23. | Kellum, R., and P. Schedl. 1991. A position-effect assay for boundaries of higher order chromosomal domains. Cell 64:941-950[Medline]. |
| 24. |
Kirillov, A.,
B. Kistler,
R. Mostoslavsky,
H. Cedar,
T. Wirth, and Y. Bergman.
1996.
A role for nuclear NF-B in B-cell-specific demethylation of the Ig locus.
Nat. Genet.
13:435-441[Medline].
|
| 25. |
Klehr, D.,
K. Maass, and J. Bode.
1991.
Scaffold-attached regions from the human interferon domain can be used to enhance the stable expression of genes under the control of various promoters.
Biochemistry
30:1264-1270[Medline].
|
| 26. | Laemmli, U. K., E. Kas, L. Poljak, and Y. Adachi. 1992. Scaffold-associated regions: cis-acting determinants of chromatin structural loops and functional domains. Curr. Opin. Genet. Dev. 2:275-285[Medline]. |
| 27. |
Levis, R.,
T. Hazelrigg, and G. M. Rubin.
1985.
Effects of genomic position on the expression of transduced copies of the white gene of Drosophila.
Science
229:558-561 |
| 28. | Levy-Wilson, B. 1995. Transcriptional control of the human apolipoprotein B gene in cell culture and in transgenic animals. Prog. Nucleic Acids Res. Mol. Biol. 50:161-190[Medline]. |
| 29. |
Levy-Wilson, B., and C. Fortier.
1989.
The limits of the DNase I-sensitive domain of the human apolipoprotein B gene coincide with the locations of chromosomal anchorage loops and define the 5' and 3' boundaries of the gene.
J. Biol. Chem.
264:21196-21204 |
| 30. |
Lichtenstein, M.,
G. Keini,
H. Cedar, and Y. Bergman.
1994.
B cell-specific demethylation: a novel role for the intronic chain enhancer sequence.
Cell
76:913-923[Medline].
|
| 31. |
McKnight, R. A.,
A. Shamay,
L. Sankaran,
R. J. Wall, and L. Hennighausen.
1992.
Matrix-attachment regions can impart position-independent regulation of a tissue-specific gene in transgenic mice.
Proc. Natl. Acad. Sci. USA
89:6943-6947 |
| 32. | McKnight, R. A., M. Spencer, R. J. Wall, and L. Hennighausen. 1996. Severe position effects imposed on a 1 kb mouse whey acidic protein gene promoter are overcome by heterologous matrix attachment regions. Mol. Reprod. Dev. 44:179-184[Medline]. |
| 33. | Mirkovitch, J., M. E. Mirault, and U. K. Laemmli. 1984. Organization of the higher-order chromatin loop: specific DNA attachment sites on nuclear scaffold. Cell 39:223-232[Medline]. |
| 34. |
Palmiter, R. D.,
E. P. Sandgren,
D. M. Koeller, and R. L. Brinster.
1993.
Distal regulatory elements from the mouse metallothionein locus stimulate gene expression in transgenic mice.
Mol. Cell. Biol.
13:5266-5275 |
| 35. | Phi-Van, L., and W. H. Stratling. 1988. The matrix attachment regions of the chicken lysozyme gene co-map with the boundaries of the chromatin domain. EMBO J. 7:655-664[Medline]. |
| 36. | Phi-Van, L., and W. H. Stratling. 1996. Dissection of the ability of the chicken lysozyme gene 5' matrix attachment region to stimulate transgene expression and to dampen position effects. Biochemistry 35:10735-10742[Medline]. |
| 37. |
Phi-Van, L.,
J. P. von Kries,
W. Ostertag, and W. H. Stratling.
1990.
The chicken lysozyme 5' matrix attachment region increases transcription from a heterologous promoter in heterologous cells and dampens position effects on the expression of transfected genes.
Mol. Cell. Biol.
10:2302-2307 |
| 38. |
Poljak, L.,
C. Seum,
T. Mattioni, and U. K. Laemmli.
1994.
SARs stimulate but do not confer position independent gene expression.
Nucleic Acids Res.
22:4386-4394 |
| 39. |
Rollini, P., and R. E. K. Fournier.
1997.
Molecular linkage of the human 1-antitrypsin and corticosteroid-binding globulin genes on chromosome 14q32.1.
Mamm. Genome
8:913-916[Medline].
|
| 39a. | Rollini, P., and R. E. K. Fournier. Unpublished results. |
| 40. | Romig, H., J. Ruff, F. O. Fackelmayer, M. S. Patil, and A. Richter. 1994. Characterisation of two intronic nuclear-matrix-attachment regions in the human DNA topoisomerase I gene. Eur. J. Biochem. 221:411-419[Medline]. |
| 41. | Roseman, R. R., V. Pirrotta, and P. K. Geyer. 1993. The su(Hw) protein insulates expression of the Drosophila melanogaster white gene from chromosomal position-effects. EMBO J. 12:435-442[Medline]. |
| 42. |
Sander, M., and T. S. Hsieh.
1985.
Drosophila topoisomerase II double-strand DNA cleavage: analysis of DNA sequence homology at the cleavage site.
Nucleic Acids Res.
13:1057-1072 |
| 43. | Schoffl, F., G. Schroder, M. Kliem, and M. Rieping. 1993. An SAR sequence containing 395 bp DNA fragment mediates enhanced, gene-dosage-correlated expression of a chimaeric heat shock gene in transgenic tobacco plants. Transgenic Res. 2:93-100[Medline]. |
| 44. |
Spradling, A. C., and G. M. Rubin.
1982.
Transposition of cloned P-elements into Drosophila germ line chromosomes.
Science
218:341-347 |
| 45. | Stief, A., D. M. Winter, W. H. Stratling, and A. E. Sippel. 1989. A nuclear DNA attachment element mediates elevated and position-independent gene activity. Nature 341:343-345[Medline]. |
| 46. | Strick, R., and U. K. Laemmli. 1995. SARs are cis DNA elements of chromosome dynamics: synthesis of a SAR repressor protein. Cell 83:1137-1148[Medline]. |
| 47. | Strissel, P. L., R. Espinosa III, J. D. Rowley, and H. Swift. 1996. Scaffold attachment regions in centromere-associated DNA. Chromosoma 105:122-133[Medline]. |
| 48. |
Talbot, D.,
P. Descombes, and U. Schibler.
1994.
The 5' flanking region of the rat LAP (C/EBP ) gene can direct high-level, position-independent, copy number-dependent expression in multiple tissues in transgenic mice.
Nucleic Acids Res.
22:756-766 |
| 49. |
Thompson, E. M.,
E. Christians,
M. G. Stinnakre, and J. P. Renard.
1994.
Scaffold attachment regions stimulate HSP70.1 expression in mouse preimplantation embryos but not in differentiated tissues.
Mol. Cell. Biol.
14:4694-4703 |
| 50. | Udvardy, A., E. Maine, and P. Schedl. 1985. The 87A7 chromomere. Identification of novel chromatin structures flanking the heat shock locus that may define the boundaries of higher order domains. J. Mol. Biol. 185:341-358[Medline]. |
| 51. |
van der Geest, A. H.,
A. H. M. Hall,
S. Spiker, and T. C. Hall.
1994.
The -phaseolin gene is flanked by matrix attachment regions.
Plant J.
6:413-423.
|
| 52. |
van der Geest, A. H., and T. C. Hall.
1997.
The -phaseolin 5' matrix attachment region acts as an enhancer facilitator.
Plant Mol. Biol.
33:553-557[Medline].
|
| 53. | Vazquez, J., and P. Schedl. 1994. Sequences required for enhancer blocking activity of scs are located within two nuclease-hypersensitive regions. EMBO J. 13:5984-5993[Medline]. |
| 54. |
Zhou, J.,
S. Barolo,
P. Szymanski, and M. Levine.
1996.
The Fab-7 element of the bithorax complex attenuates enhancer-promoter interactions in the Drosophila embryo.
Genes Dev.
10:3195-3201 |
Mol Cell Biol, April 1998, p. 2382-2391, Vol. 18, No. 4
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Silicheva, M., Golovnin, A., Pomerantseva, E., Parshikov, A., Georgiev, P., Maksimenko, O.
(2009). Drosophila mini-white model system: new insights into positive position effects and the role of transcriptional terminators and gypsy insulator in transgene shielding. Nucleic Acids Res
0: gkp877v1-gkp877
[Abstract] [Full Text] -
Gallagher, P. G., Nilson, D. G., Steiner, L. A., Maksimova, Y. D., Lin, J. Y., Bodine, D. M.
(2009). An insulator with barrier-element activity promotes {alpha}-spectrin gene expression in erythroid cells. Blood
113: 1547-1554
[Abstract] [Full Text] -
Chetverina, D., Savitskaya, E., Maksimenko, O., Melnikova, L., Zaytseva, O., Parshikov, A., Galkin, A. V., Georgiev, P.
(2008). Red flag on the white reporter: a versatile insulator abuts the white gene in Drosophila and is omnipresent in mini-white constructs. Nucleic Acids Res
36: 929-937
[Abstract] [Full Text] -
Watanabe, S., Watanabe, S., Sakamoto, N., Sato, M., Akasaka, K.
(2006). Functional analysis of the sea urchin-derived arylsulfatase (Ars)-element in mammalian cells.. GENES CELLS
11: 1009-1021
[Abstract] [Full Text] -
Namciu, S. J., Fournier, R. E. K.
(2004). Human Matrix Attachment Regions Are Necessary for the Establishment but Not the Maintenance of Transgene Insulation in Drosophila melanogaster. Mol. Cell. Biol.
24: 10236-10245
[Abstract] [Full Text] -
Weber, M., Hagege, H., Murrell, A., Brunel, C., Reik, W., Cathala, G., Forne, T.
(2003). Genomic Imprinting Controls Matrix Attachment Regions in the Igf2 Gene. Mol. Cell. Biol.
23: 8953-8959
[Abstract] [Full Text] -
Giraldo, P., Martinez, A., Regales, L., Lavado, A., Garcia-Diaz, A., Alonso, A., Busturia, A., Montoliu, L.
(2003). Functional dissection of the mouse tyrosinase locus control region identifies a new putative boundary activity. Nucleic Acids Res
31: 6290-6305
[Abstract] [Full Text] -
Byrd, K., Corces, V. G.
(2003). Visualization of chromatin domains created by the gypsy insulator of Drosophila. JCB
162: 565-574
[Abstract] [Full Text] -
Ostermeier, G. C., Liu, Z., Martins, R. P., Bharadwaj, R. R., Ellis, J., Draghici, S., Krawetz, S. A.
(2003). Nuclear matrix association of the human {beta}-globin locus utilizing a novel approach to quantitative real-time PCR. Nucleic Acids Res
31: 3257-3266
[Abstract] [Full Text] -
Ramezani, A., Hawley, T. S., Hawley, R. G.
(2003). Performance- and safety-enhanced lentiviral vectors containing the human interferon-{beta} scaffold attachment region and the chicken {beta}-globin insulator. Blood
101: 4717-4724
[Abstract] [Full Text] -
Majumder, P., Cai, H. N.
(2003). The functional analysis of insulator interactions in the Drosophila embryo. Proc. Natl. Acad. Sci. USA
100: 5223-5228
[Abstract] [Full Text] -
Donev, R., Horton, R., Beck, S., Doneva, T., Vatcheva, R., Bowen, W. R., Sheer, D.
(2003). Recruitment of Heterogeneous Nuclear Ribonucleoprotein A1 in Vivo to the LMP/TAP Region of the Major Histocompatibility Complex. J. Biol. Chem.
278: 5214-5226
[Abstract] [Full Text] -
Liebich, I., Bode, J., Reuter, I., Wingender, E.
(2002). Evaluation of sequence motifs found in scaffold/matrix-attached regions (S/MARs). Nucleic Acids Res
30: 3433-3442
[Abstract] [Full Text] -
West, A. G., Gaszner, M., Felsenfeld, G.
(2002). Insulators: many functions, many mechanisms. Genes Dev.
16: 271-288
[Full Text] -
Lemon, B., Tjian, R.
(2000). Orchestrated response: a symphony of transcription factors for gene control. Genes Dev.
14: 2551-2569
[Full Text] -
Rollini, P., Namciu, S. J., Marsden, M. D., Fournier, R. E.
(1999). Identification and characterization of nuclear matrix-attachment regions in the human serpin gene cluster at 14q32.1. Nucleic Acids Res
27: 3779-3791
[Abstract] [Full Text] -
Scott, K. C., Taubman, A. D., Geyer, P. K.
(1999). Enhancer Blocking by the Drosophila gypsy Insulator Depends Upon Insulator Anatomy and Enhancer Strength. Genetics
153: 787-798
[Abstract] [Full Text] -
Antes, T. J., Goodart, S. A., Huynh, C., Sullivan, M., Young, S. G., Levy-Wilson, B.
(2000). Identification and Characterization of a 315-Base Pair Enhancer, Located More than 55 Kilobases 5' of the Apolipoprotein B Gene, That Confers Expression in the Intestine. J. Biol. Chem.
275: 26637-26648
[Abstract] [Full Text] -
Moritz, O. L., Tam, B. M., Papermaster, D. S., Nakayama, T.
(2001). A Functional Rhodopsin-Green Fluorescent Protein Fusion Protein Localizes Correctly in Transgenic Xenopus laevis Retinal Rods and Is Expressed in a Time-dependent Pattern. J. Biol. Chem.
276: 28242-28251
[Abstract] [Full Text]
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Copyright © 2009 by the American Society for Microbiology. For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»