cifically, regulatory sequences that were implicated 
in the lymphoid-specific expression of the m gene 
were mutated in the context of the intact rearranged 
fjL gene. 
Previous studies by Dr. Philip Sharp (Massachu- 
setts Institute of Technology) and Dr. Thomas 
Kadesch (HHMI, University of Pennsylvania) impli- 
cated the E5/E2 factor-binding sites in the IgH en- 
hancer in conferring negative regulation in nonlym- 
phoid tissues. Therefore transgenic mice carrying a 
rearranged n transgene with point mutations in the 
E5/E2 factor-binding sites of the enhancer were 
generated and analyzed. The transgenic mice ex- 
pressed the E5/E2-mutated ii gene at a high level not 
only in lymphoid and muscle tissues but also in sev- 
eral nonlymphoid tissues, demonstrating the impor- 
tance of negative regulation for lymphoid-specific /x 
gene expression. The mutations of the E5/E2- 
binding sites, however, did not affect the "off" state 
of ^l gene expression in other nonlymphoid tissues. 
Possibly another tier of negative regulation is in- 
volved in actively decreasing the basal level of ex- 
pression even further. 
Regulation of the Accessibility of Nuclear 
Factor-binding Sites in Native Chromatin 
During the past two years, Dr. Grosschedl's labora- 
tory has established an assay to determine the acces- 
sibility of an individual factor-binding site in native 
chromatin and to examine its dependence on regula- 
tory DNA sequences. The assay consists of introduc- 
ing into the mouse germline a binding site for the 
prokaryotic RNA polymerase T7, alone or linked to a 
DNA fragment suspected of conferring accessibility 
in chromatin. This experimental protocol allows 
the exogenous DNA to go through the normal devel- 
opmental processes and to be inserted in the same 
chromosomal location in various cell types. Subse- 
quently, B-lineage cells (Abelson murine leukemia 
virus-transformed pre-B ceils) and fibroblastic 
cells are derived from the transgenic mice. The ac- 
cessibility of the T7 promoter residing in native 
chromatin is examined by the addition of purified 
T7 RNA polymerase to isolated nuclei or permeabil- 
ized cells and by probing for the appearance of spe- 
cific T7 transcripts. The use of the T7 promoter as a 
factor-binding site has the advantage that its recogni- 
tion by T7 RNA polymerase does not require an in- 
teraction with other proteins and allows for a simple 
readout. Analysis of pre-B cells derived from eight 
individual transgenic mouse lines carrying the T7 
promoter linked to a VP 1 reporter gene indicated 
that the T7 promoter is inaccessible in six lines. In 
two other lines, the promoter was weakly accessi- 
ble. These data suggest that the T7 promoter by it- 
self is primarily inaccessible at various chromo- 
somal locations. 
Previous experiments by Dr. Grosschedl's labora- 
tory demonstrated that the ju enhancer is necessary 
for the expression of a rearranged n transgene in 
lymphoid tissues. Moreover, experiments by others 
indicated that the m enhancer region can confer 
transcriptional activity on heterologous transgenes. 
Therefore the ^ enhancer, together with flanking 
nuclear matrix attachment regions (MARs) , was ex- 
amined for its potential to alter chromatin structure. 
Analysis of pre-B cells from seven transgenic mouse 
lines carrying the T7-VP1 gene linked to the /x en- 
hancer/MAR region indicated that the T7 promoter 
is accessible in all the lines. The number of specific 
T7 transcripts detected was proportional to the copy 
number of the transgene, suggesting that the accessi- 
bility of the T7 promoter linked to the ix enhancer/ 
MAR region is independent of its chromosomal posi- 
tion. The number of T7 transcripts obtained from 
the seven ix/MAR-Tl lines was ~ 1 0-fold higher than 
those detected with the two weakly accessible T7 
lines and at least 50-fold higher than those obtained 
from the six inaccessible T7 lines. Together these 
data indicate that the ix enhancer/MAR region can 
confer accessibility on a heterologous factor- 
binding site in native chromatin. 
Identification and Characterization of EBF 
To unravel some of the mechanisms that allow 
multiple genes to be expressed at distinct stages of 
the lymphocyte lineage. Dr. Grosschedl's laboratory 
included the promoter of the mb- 1 gene in its analy- 
sis of cell-type-specific gene regulation. The mb-1 
gene encodes a surface IgM-associated protein and 
is expressed only in pre-B cells and surface Ig- 
positive B cells but not in antibody-secreting plasma 
cells. Isolation and characterization of the mb l 
promoter in cell culture transfection assays indi- 
cated that it directs heterogeneously initiated tran- 
scription specifically in early-stage B cells. A func- 
tionally important nucleotide sequence in the 
proximal mb- 1 promoter region was shown to inter- 
act with the lymphoid/myeloid lineage-specific 
protein Pu.l. The Pu.l -binding site in the mb-1 
promoter, however, is also recognized by a ubiqui- 
tous factor, raising the question as to how the 
correct combination of these multiple factors is as- 
sembled on the mb-1 promoter. In addition. Dr. 
Grosschedl's laboratory has identified a novel pre-B 
and B cell-specific DNA-binding activity, termed 
early B cell factor (EBF), that recognizes an up- 
stream mb-1 promoter sequence. The binding site 
CELL BIOLOGY AND REGULATION 65 
