ABSTRACTS

Workshop on molecular and

Cell Biology

Sirmione, Italy, October 4, 1987

 77

OP1 OP2
USE OF TRANSFECTION TECHNIQUES FOR STUDYING GENE
THE USE OF TISSUE CULTURE OF ~BRYONIC EXPRESSION.
STE~ CELLS AS A ROUTE TO TRANSGENESIS Jacques Elion, Facult~ Xavier-Bichat, 75018 Paris,
AND INSERTIONAL MUTATION, DR MARTIN France.
EVANS, DEPARTMENT OF GENETICS.
UNIVERSITY OF CAMRIDGE, UNITED KINGDOM Transfection (or DNA-mediated gene transfer) is a
very powerful methodology for studying gene expression.
The advent of dt~eot tmoletton into First, because one uses cloned genes, their structure can
r eultume of plurtpotential ~elll be altered so as to identify cis-acting regulatory
fx~)m the eemlM moule emb~o (EK eelll) elements. Second, expression being studied in eukaryotic
has allowed ~he route s tissue cells, trans-acting regulatory factors in the cellular
culture to the whole antmel end its Eerm environment can also be identified, whether or not in
line to be s reeltmed. Wollowtns response to an extracellular stimulus. Maturation of the
senetie trenlto~mstton selection and primary transcripts can be studied. Several techniques
clontns thele EK oellm e~e able to are available to obtain high efficiency gene transfer.
~eeoloniBe a murihe embryo ahd They include calcium phosphate precipitation, use of
pe~ttotpite in the s os germ DEAE-Dextran, protoplast fusion and more recently
line ehimae~ie mice. electroporation. The choice of recipient cells and of the
plasmid vector depends upon the type of information which
One os the molt tmportent pmeetlcml is saught. The entire gene can be transfered including
dlfs between thlm route to its promoter and other putative regulatory sequences,
trmmIsenelil end that of z~Sote alternatively, a construction can be made so that the
in~ection il that the time period in gene studied is driven by a powerful heterologous
vitmo maw be uled both r allow promoter of cellular or viral origin. Finally a promoter
efs multlple cell t~enls of interest can be fused to a foreign structural gene
and to allow seleotton of Ipeotfte tame (often the bacterial chloramphenicol acetyl transferase
events, much ee mutstlon wtthln s or CAT gene). Analysis will either be aimed at the RNA
Ipeetfio Eerie ot interest. transcript (for instance by S1 mapping) or at the
resulting protein (i.e. enzymatic assay for CAT).
Semeenlns s lnlePtionel mutation both Expression of the transfered gene can be studied either
in Y.~2~2 end in vtvo will be dileulaed. transiently or after the plasmid DNA has been integrated
into the cellular chromatin to form stably transformed
cell-lines, in the latter case, the plasmid vector must
contain a dominant selection marker in addition to the
gene of interest, so as to eliminate non-transfected
cells. Retroviruses have also been used as vectors to
obtain stable transformants. The potential of these two
approaches is different and clearly a choice depends upon
the type of question to be answered.

OP3
STRUCTURE-FUNCTIONANALYSIS OF STEROID HORMONERECEPTORS
H. Gronemeyer, M. Berry, M.T. Bocquel, M. Devfc, M.V. Govindan,
S. Green, J.M. Jeltsch, Z. Krozowski, A. Krust, V. Kumar, M.E.
Meyer, G. Stack, C. Stricken, U. Stropp, B. Turcotte and P.
Chambon, LGME/CNRS and U.18q/[NSERH, Facult~ de H~decine, 67085-
STRASBOURG C~dex, France.
Our laboratory has cloned and sequenced the cDNAs corresponding
to human estrogen (hER), g]ucocorticoid and progestin receptors
end to the chicken estrogen (cER) and progestin (cPR) receptors.
Cytosolic and nuclear extracts of HeLl c e l l s ,
expressing vectors containing the d i f f e r e n t
receptors with wild-type c h a r a c t e r i s t i c s . An amino acid sequence
comparison between hER and cER revealed three highly conserved
regions A, C and E (AA 1-38, 180-263, 302-553) and three less
conserved regions B, D end F (AA 39-179, 264-301, 554-595). All
receptors and the v-erbA/thyroid receptor protein contain
sequences homologous to regions C and E. The difference in
lengths between various receptors is due to a d i f f e r e n t size of
the N-terminal h a l f (regions A and B). Transient expression in
HeLa and Cos cells of vectors containing wild type and mutated
cDNAS has been used to define protein domains responsible f o r
receptor function, i . e . ]igend binding, t i g h t nuclear binding and
t r a n s c r i p t i o n a l a c t i v a t i o n using v i t - t k - C A T , pS2-CAT or MMTV-CAT
constructs in cotransfection experiments. Region E is the steroid
binding domain, end region C which contains the putative
DNA-binding " f i n g e r s " is indispensable f o r t i g h t nuclear binding
and therefore most probable f o r DNA binding. Both regions C and E
are required f o r e f f i c i e n t a c t i v a t i o n of the hormone responsive
elements (HRE's) of the " v i t " and pS2 genes (ER) and of HHTV-LTR
(GR, PR). Regions A and B are at least p a r t l y dispensable in
these assays. A chimeric hER protein bearing the hGR region
activates the GRs of the MHTV-LTR, thus demonstrating that
hormone and target gene s p e c i f i c i t i e s reside in regions E and C.
Experiments describing the i n t e r a c t i o n of wild type and mutant
receptors with respect to the a c t i v a t i o n of target genes w i l l be
presented.
0P4
ONCOGENES A N D T H E I R E X P R E S S I O N I N B O N E
Natalie M. Teich, Janice Rowe, and Maureen Harrison, Imperial
Cancer Research Fund, Lincoln's Inn Fields, London WC2A 3PX,
England.
In the strictest sense, an oncogene is agene that causes cancer.
Nowadays one may modify this definition to those genes involved in
the carcinogenic process although perhaps without a causal role. The
genes first described generically as oncogenes were found in
retroviruses (the family of RNA-containing tumor viruses) and were
transiently shown to be responsible for a variety of acute leukemias, sarcomas
cDNAs contain and carcinomas in animals. Of particular interest was the discovery
that these oncogenes were actually "captured" (transduced) by the
viruses from their hosts; i.e., the oncogenes were originally of cellular
origin. Hence, the viral and cellular homologues were distinguished as
v-onc or c-one genes, respectively. A corollary of this finding is that
animal cells contain potential tumor genes (proto-oncogenes) within
their genomes. More than 20 different v-one genes have been
described. Some belong to multi-gene families; some share common
biochemical functions. Tumors have been analyzed for oncogene
changes and/or expression in several ways: (1) by hybridization of
tumor mRNA to oncogene DNA to assess levels of expression; (2) by
"transfection" (DNA or chromosome mediated gene transfer) of tumor
D N A into fiat, nontumorigenic cells, with morphological
transformation i_nvitro or tumorigenicity in vivo as endpoints; (3) by
analysis of chromosomal aberrations, such as translocations and
amplifications; and (4) by analysis of integration sites in
virus-induced tumors. These methods have sometimes led to the
re-identification of previously known oncogenes (i.e., from retroviral
research) and in other cases to the identification of novel oncogenes.
Identification of the tissue-specific expression, subcellular localization,
and biochemical functions of these genes has led to interesting
speculation as to their roles in normal and abnormal differentiation.
We have examined normal osteogenic cells and osteosarcoma
cells for expression of a large number of oncogenes; this study will
serve as a paradigm of gene expression in this cell lineage.
78

OP5 OP6
ONCOGENE EXPRESSION IS REGULATED BY 1,25(OH)2D 3 EFFECTS OF INSULIN-LIKE GROWTH FACTOR(IGF) I AND
IN U937 CELLS G R O W T H H O R M O N E (GH) O N G R O W T H A N D B O N E O F H Y P O X
R. Karma/i, A. Hhalla, S. Farrow, P. Lydyard AND DIABETIC RATS.
and J.L.H. O'Riordan. J. Z a p f , H . P . G u l e r , E. S c h e i w i l e r , E.R. Froesch
Middlesex Hospital, London, U.K. Metabolic Unit, Dept. of Medicine, Univ. Hospital
Expression of c-myc gene is amplified in U937 cells and C H 8 0 9 1 Z H r i c h.
may account for their abnormal proliferation. Since
1,25(OH)~D 2 inhibits proliferation of U937 cells, the IGF I stimulates replication of cultured fibre-
effect of 1,25(OH)2D 3 on c-myc gene expression was blasts and differentaition of cultured myoblasts
therefore studied and was related to changes in cell and osteoblasts in v i t r o . A s p o s t u l a t e d by the
proliferation and in cell phenotype induced by the somatomedin hypothesis, I G F I p r o m o t e s g r o w t h al-
hormone. so in v i v o as t h e m e d i a t o r o f G H a c t i o n . If t h i s
U937 cells were cultured in RPMI supplemented with 5% n o t i o n is c o r r e c t a d m i n i s t r a t i o n of IGF I in GH-
FCS in the presence or the absence of 1,25(OH)~D 3 from deficient s t a t e s s h o u l d b e a b l e to p r o m o t e g r o w t h
]5 minutes to 72 hours. Cell proliferation was assessed 300 u g / d o f I G F I i n f u s e d s c o v e r 18 d i n t o h y p o x
by [3H]thymidine uptake; cell differentiation monitored r a t s l e a d s to a s i m i l a r i n c r e a s e in b o d y w e i g h t ,
by the binding of UCHM~, which defines a monocyte-related tibial epiphyseal width, longitudinal bone growth
antigen. A dot-blot assay for c-myc mRNA was used to and new trabecular bone structures as 2 0 0 m U / d of
assess changes of oneogene expression. GH. I t c a u s e s a n e v e n g r e a t e r i n c r e a s e t h a n G H
In the presence of i,25(OH)2D 3 (Io-SM), inhibition of in t h e w e i g h t of t h e k i d n e y s , t h e s p l e e n a n d t h e
cell proliferation could only be observed after 24 hours thymus. In diabetic rats, which are GH- and IGF-
when it was decreased by 25%. By 72 hours, proliferation deficient, GH administration has no effect on
was reduced to 50% of control values. Cell differen- g r o w t h . H o w e v e r , I G F I, l i k e i n s u l i n , r e s t o r e s
tiation, as assessed by UCHMI binding, occurred earlier. body weight gain, tibial epiphyseal width, carti-
At 18 hours 45% of the cells were positive and this l a g e s t r u c t u r e o f t h e t i b i a l g r o w t h p l a t e a n d ad-
fraction increased further to 65~ at 72 hours. However jacent bone trabeculae towards normal without -
changes in c-myc mRNA levels could be ~etected even in c o n t r a s t to i n s u l i n - c o r r e c t i n g the metabolio
earlier. These were reduced by 47% of control values at derangement. Conclusions: IGF I stimulates organ
6 hours with a further decrease by 70% at 24 hours. It a n d b o n e g r o w t h i n v i v o i n t h e a b s e n c e o f GH. It
then remained at the same level up to 72 hours. e x e r t s t h e s e e f f e c t s in a n e n d o c r i n e f a s h i o n . In
Thus the effect of 1,25(OH)2D 3 on U937 cell involves c o n t r a s t to I G F I, GH is n o t a n a n a b o l i c h o r m o n e
an early decrease of c-myc transcription, which precedes in t h e a b s e n c e o f i n s u l i n . I n s u l i n is l i k e l y to
phenotypic changes and reduction in DNA synthesis. normalize g r o w t h in d i a b e t i c r a t s v i a I G F I b y
restoring GH secretion and the responsiveness of
the l i v e r t o p r o d u c e I G F I u n d e r t h e i n f l u e n c e
of GH. A l l o f t h e s e f i n d i n g s a r e c o n s i s t e n t w i t h
the somatomedin hypothesis.

OP7 OP8

VOLTAGE ACTIVATED IONIC CHANNELS AND CONDUCTANCES IN EXPREssION OF CALcITONIN/CGRP-I and - I I GENE PRODUCTS IN
CULTURED 0STEOBLASTS HUMAN TUMOR CELL LINES. W. Born, S. Haller-Brem, R. Muff,
R. R i z z o l i , J.-Ph. Bonjour anTn-d--J.A. Fischer. Research La-
D.L. YPEY (i), J.H. RAVESLOOT (l,Z). P.J. NIJWEIDE boratory f o r Calcium Metabolism, U n i v e r s i t y of Zurich,
(2[. H.P. BUISMAN (i) 8008 Zurich, Switzerland, Di vi si on o f Pathophysiology,
Department o f Medicine, U n i v e r s i t y H o s p i t a l , Geneva,
Patch-clamp measurements were made on osteoblast-like Switzerland.
cells isolated from embryonic chick calvaria.
Cell-attached-patch measurements revealed two types of Two s t r u c t u r a l l y r e l a t e d c a l c i t o n i n ( C T ) / c a l c i t o n i n gene-
depolarization-activated high conductance (100-250 pS) r e l a t e d peptide (CGRP) genes-I and - I I have been i d e n t i -
channels. One type showed no or only weak f i e d in man (Steenbergh et a l . : FEBS L e t t . 209:97, 1986).
inactivation. The second type exhibited slow hut Part o f the CGRP-II gene represents a pseudogene f o r a CT-
almost complete inactivation during 10 s depolarizing l i k e peptide which is not expressed.
voltage pulses. The single channel conductances of We have characterized mRNA from the CT/CGRP-I and the CGRP
these channel types were about i00 pS or 250 pS, - I I genes in human tumor c e l l l i n e s using Northern bl ot s
depending on whether the pipettes were filled with a 3 with s p e c i f i c antisense RNA probes. The peptides encoded
mM K+ or 143 m M K + saline, respectively. by the genes were analyzed by s p e c i f i c RIAs in combination
Whole-cell measurements revealed the existence of two with peptide separation methodology. Mature CGRP-I mRNA is
types of outward rectifying conductances. The first a predominant product o f the two genes in a medullary thy-
type conducts K+ ions and activates within 20-200 ms roid carcinoma (TT) c e l l l i n e . The c e l l e x t r a c t s contain
upon depolarizing voltage steps from -60 mV to -30 mV mature CGRP-I and CT. Both peptides were also released i n -
or higher. It inactivates almost completely with a to tissue c u l t u r e medium together with CGRP-I and CT pre-
time constant of 2-3 s. Recovery from inactivation is cursor p r o t e i n s , and a large mol wt CGRP-II component. In
biphasic with an initial rapid phase (1-2 s) followed a lung tumor (BEN) c e l l l i n e , CT mRNA exceeded CGRP-I mRNA.
by a slow phase (> 20 s). The second whole-cell While precursors o f CGRP-I and CT were detected, the ma-
conductance rapidly activates upon voltage steps to ture peptides were not recognized. In a Ewing sarcoma c e l l
membrane potentials of > +50 mV, but inactivates only l i n e , CGRP-II s p e c i f i c t r a n s c r i p t s were only detected. A
weakly. Its single channels of 140 pS conductance CGRP-II p r o t e i n was present, but the mature peptide was
were not highly K+, CI- or Na+ selective. not i d e n t i f i e d .
We hypothesize a role for such ionic channels in In conclusion, v a r i a b l e expression and maturation o f CT/
mineral metabolism of bone tissue and its control by CGRP-I and CGRP-II gene derived pr ot ei ns was observed in
osteoblasts. three d i f f e r e n t human tumor c e l l l i n e s . Relative amounts
of mature CGRP-I and CT mRNA in the TT c e l l l i n e r e f l e c t e d
(i) Dept. of Physiology and (Z) Dept. of Cell the l e v e l s o f CGRP-I and CT peptides. In the Ewing sarcoma
Biology and Histology. University of Leiden, The c e l l l i n e , the CT/CGRP-I gene was presumably s i l e n t . A
Netherlands. CGRP-II gene derived product was i d e n t i f i e d as a large mol
wt p r o t e i n .

OP9
A T~IRD CALCITONIN RELATED SEQUENCE IN THE HUMAN G-ENOME.
N. Alevizaki, F.V. Rassool, K.L.S. Collyear, I. MacIntyre &
8. Legon. Dept of Chemical Pathology, Royal Postgraduate
Medical School; London, England.
The study of the calcitonin (CT) gene in both rat and
man revealed the existence of a previously unidentified al-
ternative peptide product of the gene, CT gene related pep-
tide (CGRP). Further studies showed that there was a second
gene, the beta gene, encoding another CGRP-Iike molecule.
We have shown that the beta CGRP gene also contains a CT-
related sequence diverging by 35% from the alpha gene. Al-
though this has the potential to code for a CT-like pepti-
de, it is probably not expressed in man.
However these two genes are not the only CT-related
genes in the human genome. We have recently isolated a
third (gamma) human genomic sequence with a very close homo
Jogy (90%) with exons II and III which code for the amino-
terminal peptide that CT and CGRP share within their pre-
Cursors. As detection of a salmon CT-like molecule has been
repeatedly reported in mans the possibility that this is
encoded by this genomic locus has been investigated. Two
overlapping genomic clones spanning an area of 20 kb around
this sequence were anlyeed using modified hybridisation
conditions which should allow cross-hybridisation between
two sequences whose encoded peptides differ by about 50%.
No CT or CGRP related sequences were detected in our clones.
Furthermore, sequence analysis of the amino-terminal pepti-
de related region indicates that this has probably been de-
>ived from the beta gene as the whole of excn 2- intron 2-
exon 3 region is conserved to the same degree. Using in si~u
hybridisation we have localised this sequence to the short
arm of chromosome ii. These data suggest that it is proba-
bly the result of a relatively recent duplication.
We suggest that if a salmon-like CT gene exists in the
human genome at all, it is very unlikely that it is asso-
ciated with the gamma sequence.
OPII
M O L E C U L A R CLONING, EXPRESSION, AND A P P L I C A T I O N OF cDNA
E N C O D I N G THE V I T A M I N D RECEPTOR. J. W e s l e y Pike. Baylor
College of Medicine, Houston, Texas USA.
V i t a m i n D receptors (VDR) are i n t r a c e l l u l a r trans-
cription factors that m e d i a t e the h o r m o n a l a c t i o n of 1,25-
d i h y d r o x y v i t a m i n D 3 (1,25(OH)2D3). The recent m o l e c u l a r
cloning of these proteins from chicken, rat, and human
sources has r e v e a l e d a common s t r u c t u r a l a r c h i t e c t u r e
comprised of two distinct domains whose p r i m a r y sequences
are v i r t u a l l y c o n s e r v e d from species to species, and w h i c h
show a striking degree of h o m o l o g y w i t h similar domains
found w i t h i n other m e m b e r s of the steroid receptor gene
family. Two cDNAs r e p r e s e n t i n g the entire coding region
of human V D R were ligated at a c o m m o n r e s t r i c t i o n site and
cloned into an a d e n o v i r u s m a j o r late p r o m o t e r - d r i v e n
m a m m a l i a n e x p r e s s i o n v e c t o r (p91023b). Acute t r a n s f e c t i o n
of this construct into COS-1 cells r e s u l t e d in high expres-
sion ( 250,000 c o p i e s / c e l l - a v e r a g e ) of a recombinant pro-
tein d i s p l a y i n g w i l d t y p e c h a r a c t e r i s t i c s of m o l e c u l a r mass
52,000, an e q u i l i b r i u m d i s s o c i a t i o n constant (K d) for 1,25
(OH)2D 3 of 5x10-11M, and a b i l i t y to interact with immobil-
ized DNA. E x p r e s N i o n and assay of a series of 3' V D R
cDNA deletion m u t a t i o n s reveal that D N A b i n d i n g a c t i v i t y
resides in N - t e r m i n a l domain I and s t e r o i d - b i n d i n g is
a s s o c i a t e d w i t h C - t e r m i n a l domain II--the f u n c t i o n a l char-
acteristics of these domains c o r r e s p o n d to similar regions
found in other steroid receptors. Several human v i t a m i n
D - r e g u l a t e d target genes are c u r r e n t l y being d e v e l o p e d in
which to test and localize the t r a n s c r i p t i o n a l a c t i v i t y of
recombinant VDR. VDR cDNA probes are p r e s e n t l y being ex-
ploited to define the s t r u c t u r a l o r g a n i z a t i o n of the
n a t u r a l human VDR gene and to identify its unique promoter
elements. Simultaneously, these probes are being u t i l i z e d
to evaluate the genetic basis for the resistance syndrome
v i t a m i n D - d e p e n d e n t rickets, type 2. Both p h y s i c a l and
functional evidence has a c c u m u l a t e d to implicate aberrant
VDR genes as the u n d e r l y i n g cause of this disease. Fur-
ther a p p l i c a t i o n of VDR gene probes should prove
essential in defining the p h y s i o l o g y and p a t h o p h y s i o l o g y
of v i t a m i n D action.
79
OPIO
CALCIUM ADMINISTRATION PROVOKES TRANSCRIPTIONAL
AND POST-TRANSCRIPTIONAL MODIFICATIONS OF CALCI-
TONIN mRNA IN THE RAT.
Marie-Christine Delehaye, Nadine Segond, Jacqueline
Taboulet, G~rard Milhaud, Mohsen S. goukhtar and Annick
Jullienne.
INSERM Ul13 and CNRS UA 163, Laboratoire d'Endocrinologie
Mol~culaire, CHU St. Antoine, 27 Roe Chaligny, 75571 Paris cedex 12,
Calcium, administered in rats, stimulates the secretion of
calcitonin (CT). We have previously shown that calcium elicits a rapid
increase in translatable calcitonin mRNA. in this work, plasma and
thyroid glands were obtained at 2, 30, 60, 120 and 240 minutes after
Ca injection and CT levels estimated by a specific radioimmunoassay for.
the hormone. Poly A rich RNA were extracted from thyroids and
purified by oligo dT cellulose, and CT mRNA quantified by its ability to
direct the synthesis of calcitonin (CT) precursors in a cell free system
and by hybridization to a 32p cDNA probe specific for CTmRNA. We
report here that the levels of hybridizable CT mRNA remained constan~
up to 30 minutes and then increased from 1 to 4 hours. Translational
activity of CT mRNA varied during the experiment: a sharp and
transient rise, as previously reported, occured at 2 minutes and a:
second increase after 30 minutes. CT plasma levels reached peak values
at 2 minutes and then declined rapidly, reaching basal levels at four
hours. CT tissue contents decreased significantly, two minutes after the
injection, normal levels were recovered by 30 minutes and remained
unchanged to 4 hours. This suggests that the actions of Ca ions are
complex, as the ion exerts a dual action on calcitonin mRNA levels in the
thyroid gland: an early post-transcriptional rise only in translatable
calcitonin mRNA, presumably by the activation of inert mRNA pools and
a late transcriptional increase in both translatable and total poly A +
calcitonin RNA. This action results in the biosynthesis of new hormone
and thus to a restoration of tissue levels of calcitonin. Subsequently the
pools of calcitonin mRNA are restored by an increase in the
transcription rate.
OPI2
ZTRUCTURAL ANALYSIS OF THE GENE FOR RAT VITAMIN D-DEPENDENT
CALCIUM-BINDING PROTEIN (gK CaBP).
C. PERRET 7 N. LOMRI, N. GOUHIER, C. AUFFRAY*, M. THOMASSET.
INSERM U.120, Alli6e CNRS 78110 Le V@sinet and *CNRS Insti-
tut d'Embryologie Nogent/Marne-France.
The structural organization of the entire rat 9KCaBP (oho
lecalcin or oalbindin) gene was determined by analysis of
overlapping genomic clone s isolated from a rat genomic li-
brary using the rat 9KCaBP cDNA (Desplan et al J. Biol.
Chem. 258, 13502, 1983). These c l o n e s t o g e t h e r span 30 kbp
of rat genomic DNA with the rat 9KCaBP gene lying in the
middle. The 9KCaBP gene is 2 . 5 kbp long and contains 3 e-
xons interrupted by 2 introns. The first exon contains al-
most the entire 5' untranslated region. The second exon,
which is separated from the first by one intron of 308 bp,
contains the end of the 5' untranslated region and codes
for the calcium-binding site I. The third exon codes for
the calcium binding site II and the 3' untranslated region.
A 1806 bp intron separates the two last exons. Therefore
each of the calcium-binding domains is encooed by single,
separate exons. The transcription initiation Rite was iden-
tified by SI nuolease mapping and primer extension. A con-
sensus sequence TATAAA was localized 31 bp upstream from
the cap site and a "CAAT-box" (GAAAG) lies 72 bp upstream
from the transcription start. One _(A8)25 and one ._(AG)2 re-
. 3
peats are present in the second lntron together with an Alu~
Like sequence. Repetitive elements are present 5 kbp ups-
tream from the cap site and in the 3' flanking region. C o m -
parison of the known rat CagP sequences (9KCaBP, 28KCaBP,
$ nn protein) shows that the 9KCaBP is more closely r e l a t e d
t~V~he S protein than to the 28KCaBP. There is no evi-
i00
denoe to indicate that 9KCaBP has arisen from the 28KCaBP.
Supported in part by CNRS, biologie mol~culaire du g~ne
033107.
80

OPI3
STRUCTURAL ANALYSIS OF THE 5'-END REGION OF THE GENE ENCO-
DING THE VITAMIN D-DEPENDENT CALCIUM BINDING PROTEIN
(CALBINDIN D28K) OF THE CHICK
S. Ferrari, E. Drusiani, M. Fregni.
Istituto di Chimica Biologica, UniversitA di Modena, Via
Capi 287, 41100 Modena, Italia.
A genomic library was constructed by inserting partial
SamHI digests of chick DNA (20 kb fragments in the average)
into the bacteriophage ~ENBL3 DNA. To improve the possibi-
lity of selecting clones which contain the 5'-end region
and flanks of the Calbindin D28K gene, the library Was
screened with a synthetic oligonucleotide representing the
sequence coding for the first i0 aminoacids of the first
putative calcium binding domain.
After screening about 106 recombinants, we isolated two
overlapping clones, CBAI and CBCI, which contain inserts of
about 12kb and 18kb respectively. Appropriate restriction
fragments were subeloned in plasmid vectors suitable for
sequence analysis by the dideoxy chain termination method.
Sequencing data so far available allow to make the fol-
lowing observations.
i. The first nucleotide transcribed in the mRNA sequence
(i) occurs at position 448 in the CBAI sequence. The mRNA
sequence is colinear with the gene until nucleotide 185;
at this point the first intron (272 nucleotides in length)
occurs, followed by a short stretch of coding sequence
(from position 186 to position 262). Then a second intron
of about 1.5kb is found, followed by further coding sequen-
ce up to nucleotide 336 and a third intron.
2. The analysis of the DNA region located 5' to the first
transcribed nucleotide, allows the identification of se-
quence elements commonly found in promoters, as a "TATA
box" and several "GGCGGG boxes".
3. Clone CBCI extends CBAI by about 4kb, which are located
at the 5'-end side, with respect to mRNA polarity.
Studies are presently undertaken to establish the acti-
vity and the sensitiviy to 1,25(OH)2D 3 of the putative
promoter region in transfected cells.
i. Hunziker W. Proc Natl Acad Sci USA 83, 7578-75823 1986.
Supported by the Progetto Finalizzato Ingegneria Genetica
of the C.N.R.
OPI4
CALBINDIN D28 HAS SIX EF HAND-LIKE DOMAINS AND IS VERY
CONSERVED IN EVOLUTION
Willi Hunziker and Solveig Schrickel, Central Research
Units,
F. Hoffmann-La Roche & CO. Ltd., CH-4002 Basel
Switzerland
Chicken and rat Calbindin D28 cDNA clones were isolated
from an intestinal and a brain library respectively.
The open reading frame in both cases codes for a
protein containing six repeats of a domain with the
general property of an EF hand calcium binding site. In
both proteins within domains II and VI two and three of
the five oxygen containing amino acids important for
the coordination of calcium are replaced. Therefore
these two domains have most likely lost their calcium
binding ability. There is a moderate amino acid
homology between the six domains within the protein
(10-39%) in both cases, showing that the duplication
event that gave rise to the six domain structure
occurred early in evolution. Additionally this moderate
homology also shows that maintaining a calcium binding
EF hand domain is a weak conservation pressure. This
conclusion is further supported by the lack of an
appreciable homology of Calbindin D28 with other
calcium binding proteins. Comparing the Calbindin D28
sequence between chicken and rat there is a 79% amino
acid homologY. Considering radical amino acid changes
as differences only, the homology increases to 95% in
spite of the 300 million years since divergence of the
two species in evolution. Since maintenance of a calcum
binding site seems to be a weak conservation pressure,
additional physiological functions of Calbindin D28
have to be postulated. Calcium dependent interactions
with other cellular components could require a very
precise structure of the protein that would explain the
observed conservation in evolution.

OPI5
THE sTRUcTuRE OF THE CALBINDIN GENE
D.E.M. Lawson, P.W. Wilson, M. Harding
AFRC ~ A n i m a l Physiology and Genetics
Cambridge, England

The s t r u c t u r e of the chick c a l b i n d i n gene has been

s t u d i e d using two overlapping clones i s o l a t e d from a
c h i c k gene l i b r a r y . The gene was a n a l y s e d by cDNA
probing of r e s t r i c t i o n fragments and by nucleotide
sequencing. The c l o n e s c o n t a i n e d between them over
20Kb of chick DNA with the c a l b i n d i n gene coding f o r
the l a r g e s t mRNA accounting f o r about 18Kb. Calbindin
i s encoded f o r by at l e a s t 11 exons. The smallest is
50Kb and the l a r g e s t is the untranslated region of the
mRNA of 2500b. Three polyadenylation signals have been
i d e n t i f i e d e x p l a i n i n g the presence of the three forms
o f c a l b i n d i n mRNA, The c a l b i n d i n gene is s i m i l a r to
genes of other members of the troponin C superfamily in
t h a t the exons do not encode a functional domain in the
p r o t e i n but the p o s i t i o n of the exons d i f f e r w i t h i n the
protein sequence. The gene arose by successive
a d d i t i o n s o f the gene encoding the a n c e s t r a l two Ca
b i n d i n g domain p r o t e i n . The organisation of the
c a l b i n d i n gene w i l l be discussed. The clones contain
about 760b o f 5' upstream sequences w i t h a p o s s i b l e
promoter sequence ATAAAT a t -32b and a p o s s i b l e RNA
polymerase t r a n s c r i p t i o n factor SPI binding site
beginning at -109b from the deduced cap s i t e .
T r a n s f e c t i o n studies to t e s t these p o s s i b i l i t i e s are
being c a r r i e d out.
 81

R1 P2

HUMAN MONOMERIC CALCITONIN (hCT-m), MIDDLE-MOLECULE THE MITOGENIC EFFECT ON CHICKEN CHONDROCYTES OF
PARATHYROID HORMONE CAN BE SEPARATED FROM RENAL
PARATHGRMONE (PTH), 0STE0CALCIN dOT) AND CALCIUM-PHOSPHORUS
ADENYLCYCLASE ACTIVATING~AND RECEPTOR-BINDING-REGION.
METAZOLISM IN PATIENTS WITH MULTIPLE MYELOMA. H.Mayer, K.-D. Schl~ter, E. Wingender
A.Scazzoso, E.Cunietti, V.Righini, R.Guzzetti, R.Gandini,
G.Norbiatoand M.Bevilacqua. The mitogenic effect of parathyroid hormone (PTB) on
Ospedale L. Sacco e V. Buzz] Mllano ITALY chmndrocytes, which were established as primary cultures,
was studied by assaying the 3H-methyl-thymidine-
incorporation into DNA. Chondrocytes were isolated from
Bone destruction is a common feature of multiple myeloma sterna of 16 day old chicken embryos by collagenase
(MM).There are however few data about the possible secondary treatment and were plated in FCS poor medium on micro--
involvement of hormones regulating calcium metabolism. We titerplates. Optimal assay conditions were determined by
studied 36 pa~.ients (age range: 40-86 years) with secretory varying cell age, plating density and incubation time.
MM; 15 patients were free of osteolytie lesions (by total Under these conditions DNA synthesis was significantly
stimulated by PTH and some of its fragments. Compared to
body scitntigraphy) and 21 had bone lesions. Data as
control cultures, cells treated with hPTH(I-84) bPTH(I-34)
follows: and bPTH(3-34)-NNT displayed 2.5-fold 3H-methylthymidine
incorporation in a dose dependent manner. However,
Patients without bone destructions: hPTH(28-48) lead to a stimulation by a factor of 6,
Ca(S) P(S) Ca(U) P(U) PTU hCT OT OHP(U) whereas hPTH(43-68)T and hPTH(52-84) had no effect.From
our results obtained with overlapping PTB-fragments we
mg/dl mg/dl mg/24h mg/24h pmol/L pg/ml ng/ml mg/24h
conclude a mitogenie core region located at position
mean 9.8 8.7 153.0 869.1 67.3 4.7 6.4 19.2 28-34. It is known that the 28-48 fragment and the 28-34
SE 0.i 0.2 37.6 187.4 3.8 0.4 1.2 3.8 fragment have no demonstrable binding on the renal
Patients with bone destructions: receptor. The 28-48 region is however recognised by
mean 9.8 8.3 129.2 547.6 62.3 9.9 e 8.P } 34.3 ~ enzyme(s) that metabolize the PTH. The two N-terminal ~A
8E 0.2 0.2 20.3 74.a 4.8 1.6 1.0 6.5 which are the determinants for activation of the renal
~P O. 05 ad6nylcyclase, are not required for the cell
proliferative effect. Fusionprotein expressed in E.eoli,
representing N-terminal extended hPTH, which does not
A significant relationship was found between urinary activate the renal adenylcyelase, also stimulated DNA-
hydroxiproline and serum osteocalcin (r =0.61; P O.Ol),In synthesis.
conclusion: i) the greater bone involvement in patient with The finding that the mitogenic proliferation on
bone disease increases hCT-m in the absence of chondrocytes can occur withou~ hormone binding and
action in the renal adenylcyc ase system provides a new
!~perealcemia, suggesting some other factor controls hCT-m;
insight in the important role of PTH on bone volume
2) the activation of the osteoclasts in patients with
regulation.
multiple myeloma and bone-lesions is followed by an increase
o f osteocalein suggesting an enhanced bone-turnover.

P3
THE INFLUENCE OF PHYSICAL ACTIVITY ON THE REGULATION OF
CALCIOTROPHIC HORMONES AND THE METABOLISM OF CALCIUM AND
PHOSPHORUS IN YOUNG FWkTS. J.K. Yeh and J.F. Aloia,
Winthrop-University Hospital, Mineola, N.Y., U.S.A.
Exercise enhances bone formation and immobilization
enhances bone resorption. The current study assessed the
response of calciotrophic hormones, serum and urine
chemistries to exercise training and ilmi~obilization in
the rats.
Sixty female Spragae-Dawley rats (i00 gm +_ 5 body
weight) were divided into 3 groups. Group i consisted of
the following: exercise with pair feedinq to control
levels (EX-P). '['he treadmill speed was 25 meter per
minute for one hour/day and 5 days/week. Group 2
consisted of rats i~mobilized by sciatic denervation (IMB)
and group 3 was an untreated control group (CON). Nine
weeks exercise traininq resu]ted in a significant decrease
in sert~n phosphate (6.9+.09 vs 7.6+.15 mg/d], P<.01),
nephroqenous C-AMP (].73+.13 vs 3.67+.19 n mole/dl GE,
P < .01) and increased serum 1.25-dihydroxyv]tamin D
(71.4+3.8 vs 52.9+6.2 pg/ml, P<.05), alkaline phosphatase
(89.5+6.25 vs 70.~+4.5 IU/L, F < .05) and urinary
hydrox-fprolJne (730,56 vs 540~68 ug/day, P<.05).
Immobilization resulted in a decrease in nephrogenous
C-AMP (1.43+.]9 vs 3.87}.15 n laole/dl GF, P<.01) and
increase in--serum phosp]Late (8.0+.07 vs 7.6+.15 mg/dl,
P<.05), alkaline phosphatase (i02+i].5 vs 7~.2~4.5 IU/I,
P<.01) , urinary hydroxyproline (880~56 vs 540+68 ug/day,
P<.0]), and calcium (3.43+.45 vs 2.~2 +.3 mg/day, P<.05).
Overnight fasting resulted in a decrease in serum calcium
and increase ill parathyroid hormone in the EX group and
no significant change in the ]MB and CON groups.
Exercise enhances bone turnover with a greater
increase in formation than resorption. All increase Jn
the demand for minerals may result in increased levels of
1,25-dJhydroxyvitam~n D and intestinal absorption. IMB
enhances bone turnover with more resorption than for-
marion. Bone resorption may increase serum levels of
c;]]cJlml and phosphal-r., c~oeyoa%o paYa~h'/roJd }loymnn<,s,
resulting in increased urinary excretion of calcium.
P4
OSTEOBLASTIC EXPRESSION OF MARROW STROMAL DERIVED CELL
LINE; IN-VITRO AND IN-VIVO.
D._Bena~ahu I, Y. Kletter l, D.Zipori 2 and S. Wientroub 1'3.
Sackler School of Medicine, Tel-Aviv University I,
W e i z m a n n Institute of Science 2 and Tel-Aviv Medical
center 3, Tel-Aviv, Israel.
Bone marrow derived osteoblast~e cell line was estab-
lished by maintaining dense cultures of SJL/J mouse marrow
stromal cells. Cultures~ weekly fed, were grown in DMEM
and 10~o FCS, without passaging up to i0 months (Zipori et.
al, 1984 ) . MBA-15 one of the cell line derived, exhibited
phenotype characteristics of osteoblastic expression:
_AA. histoehemical examination demonstrated that more then
70~o of the cells were stained positively for alkaline
phosphatase (AP). B. high levels of AP activity was
measured biochemically and were enhanced 3 folds by
100nM/ml deoxamethasone. C. gel electrophoresis of
3H-proline radiolabeled cultures showed that biosyntheti-
cally these cells produce solely type I collagen. D. The
cells were responsive to the 1-34 fragment of human para-
thyroid hormone as indicated by 50 to 60 folds increase
in intracellular cAMP. E. PGE2~ but not caleitonin,
stimulates cAMP accumulation up to 30 to 60 folds. F. the
cells formed a thick, extraeellular matrix that minere-
lized in-vitro when provided with ascorbic acid and
glycerophosphate. G. hydroxyapatite crystals were
detected in-vitro using transmission electron microscopy
as well as electron diffraction analysis. H. MBA.15 cell
line produce bone in diffusion chambers implanted for 6-8
wks. in the peritoneal cavity of nude mice.
These results indicate that MBA-15 cells posses osteo-
blastic characteristics in-vitro and osteogenic potential
in-vivo.
82

P5 P6
DISCREPANCY BETWEEN TYPICAL Ca2+-INDUCED ULTRA-
INFLUENCE OF DECALCIFICATION ON THE IMMUNO- STRUCTURAL ALTERATIONS AND DEPRESSED CALCIUM
REACTIVITY OF CELLULAR ANTIGENS CONTENT, IN S K E L E T A L MUSCLES FROM VITAMIN D-
Nicholas A. Athanasou, Julian Quinn, Colin G. Woods1 DEFICIENT RATS.
and James O'D McGee R.Toury, Y.Dupuis, E.Boissonneau and N.Stelly
Nuffield Departlnent of Pathol(lgy, U n i v e r s i t y of Oxford, Lab. Biol. Cel. CNRS 94200 IVRY-sur-SEINE France
John R a d c l i f f e H o s p i t a l , and Nuffield O r t h o p a e d i c Lab. Metab. Mine. EPHE, Fac. Pharmacie, 92290
(;entre, Oxford CHATENEY-MALABRY, France.

Skeletal muscles of v i t a m i n D - d e f i c i e n t rats

ImmunohistochemicaI a n a l y s i s of s u r g i c a l s p e c i m e n s
using monoclonal a n t i b u d i e s has come to p l a y an showed the characteristic Ca2~induced damage
i m p o r t a n t r o l e in h i s t o p a t h o l o g i c a l d i a g n o s i s . Although genarally attributed to t h e s t i m u l a t i o n of
r o u t i n e f o r m a l i n f i x a t i o n and p a r a f f i n embedding soluble neutral proteases a n d of p h o s p h o l i p a s e s
halm been found to i n t e r f e r e only p a r t i a l l y with by the cation; massive regression of m y o f i b r i l s
a n t i b o d y s t a i n i n g of many useful c e l l u l a r components, sarcoplasmic reticulum, glycogen and mitochon-
the effect of v a r i o u s r e c o g n i z e d d e c a l c i f i c a t i o n r e g i m e s dria. Moreover, Z-disk weakening and defective
on the i m m u n o r e a c t i v i t y of t h e s e antigene i s p o o r l y thick myosin filament relaxation were observed
defined. We have examined the e f f e c t s of, s e v e r a l in a n u m b e r of s a r c o m e r e s . Thick filaments,
d e c a l c i f y i n g agents i n c l u d i n g strong a c i d s (5% ftgl, either individually o r in t h e i r totality,
5*d tINO3), a p r o p r i e t a r y d e c a l c i f i e r (RDO), 590 remained attached to t h e Z - d i s k s after apparent-
t r i c h l o f f a c e t i c a c i d (TCA), weak a c i d s (10% formic a c i d , ly n o r m a l contraction in t h e i r sarcomeres. The
10% a c e t i c a c i d ) , IIC1/EDTA, EDTA on the immune- altered activities of t h e C a 2 + - s e n s l t i v e enzymes
t~istochemical s t a i n i n g of f i x e d c a l c i f i e d and u n c a l c i f i e d :malate-dehydrogenase and isocitrate-dehydrogena
normal and p a t h o l o g i c a l s u r g i c a I s p e c i m e n s . The se in i s o l a t e d cytosol, corroborated the enhan-
cement of i n t r a m u s c u l a r calcium. In c o n t r a s t ,
results were compared with ialmunostaining of
measurements of t o t a l calcium by a t o m i c absorp-
undecalcified tissue from the same specimens. Even
decalcification in strong acids preserved the imnmno- tion spectroscopy disclosed reduced amounts of
the metal in h o m o g e n a t e s as w e l l as in i s o l a t e d
reactivity of many useful cellular antigens, including
subcellular ~ractions of D - d e f i c i e n t muscles.
l e u c o c y t e common antigen, i n t e r m e d i a t e filament
components, $100 p r o t e i n , p r o s t a t e , t h y r o i d and It is c o n c l u d e d that:
melanoma a s s o c i a t e d a n t i g e n s , immunoglobulins and 1 ~ vitamin D-deficiency results in d e c r e a s e d
tlI,A-DR, a l t h o u g h m o r p h o l o g i c a l and s t a i n i n g q u a l i t y was total intramuscular calcium, but increased
r e d u c e d . Weak a c i d s , EDTA and more r a p i d d e c a l c i f i e r s , available Ca2+;
TCA and IIC1/EDTA pr oduced b e t t e r m o r p h o l o g i c a l d e t a i l 2 ~ vitamin D seems induce the synthesis, or
and s t a i n i n g q u a l i t y in t i s s u e s e c t i o n s with p i ' o s e r v a t i o n stimulate the activity, of an i n t r a m u s c u l a r Ca 2+
of the i m m u n o r e a c t i v i t y of a w i d e r range of a n t i g e n i c buffer mechanism,
components. Knowledge of t h e p r e s e r v a t i o n of antigen
r e a c i i ' v i t y in d e c a l c i f i e d t i s s u e w i l l be useful in bone
r u s e a r c h and t~istopathologiea] d i a g n o s i s .

P7 P8

INCREASED LEVEL OF CALCITONIN m R N A AFTER ACUTE POTASSIUM CALCITONIN LIKE-IMMUNOREACTIVITYAND CALCITONIN GENE
&DMINISTRATION IN T H E RAT. J.-M. Garel, V. Jousset, P. Bee- E• IN RAT TISSUES. V. JouSS~, B. Leg~nd~e, P. B~s-
nard, N. Segond ~ and A. Jullienne ~ Endocrinologie M o l 6 c u - nard, N. Segond ~ A. Jullienne ~ and J.-M ~. Gar~.
laire et M6tabolique, U.P.M.C.,Paris and o U 113 de I'INSE- Endoerinologie Mol~cula~e et M~tabolique , U. P.M. C. , P ~
RM, Paris. and o U 113 de l'INSERM, Paris.

We have investigated the acute effects of elevated plas-
ma potassium concentrations on the CT m R N A leve| m~asured
by dot-blot hybridization. Plasma CT levels were signifi-
cantly increased (x2) 30 min. after potassium administra-
tion (1.2 m m o l . / 1 0 0 g bw) in adult female rats ; a trend
towards increased values was observed 10 min. after treat-
ment. No change in plasma calcium concentrations was indu-
ced b y ~ h e elevated extracellular potassium levels.
The ~ P - l a b e l e d cDNA probe was the Pstl-cleaved insert
of pBR 3271 (Le Moullec et ai.,1984) digested with Sstl to
remove the terminal poly(A) extension. The insert was fur-
ther cleaved with Bglll to obtain a probe specific of the
coding sequence of human CT. In the two experiments which
were done at 6 months intervals, %he amount of CT m R N A
measured by hybridization was significantly increased 10
min. and 30 min. (around 40 to 50%) after potassium treat-
ment. These finfings demonstrate for the first time that
potassium stimulate CT secretion by a mechanism involving
a rise in the CT mRNA level. Since potassium administration
is probably associated with an increase of cytoplasmic free
calcium concentration of the C cell, our results suggest
that calcium ion acts at a transcriptional level may be
through the calcium activation of protein(s) interacting
with DNA.
Recently, the pr~ence of monomerie cal~Ltonin (CT) in
plasma and milk wa~ reported in a l a c t ~ n g woman sw~gica-
l l y thyroidectomized (Berg e t a & , 1987). Since such findin-
gs were b~ed on immunologi~-~---a~gamen~ we have investiga-
ted the CT gene expression in .'cat mammary gland and in rat
placenta.
CT mRNA were d ~ e ~ e d by dot-blot h y ~ d i z ~ o n of t o t a l
RNA e~t~a~ed from rat t ~ s u e s with a JLp-labeled cDNA pro~
be. The PstI-~eaved i n s e r t of pBR 3271 (Le Moullec et a l . ,
1984) wa~ ~ t e d w~h SstI to remove the poly(A) exten-
sion, and the i n s e r t was further ~eaved with BglII t~ obt-
ain a probe spe.cific of the coding sequence of h~an CT.
Subcell~ f r a ~ o n s of each t ~ s u e were screen for CT-li-
ke immunorea~tivity using ~ o d i f f e r e n t a~tibodi~.
With one antibody, CT-like ~ u n o r e a ~ i v i t y was dete~ed
both in placenta and mammary gland. I m ~ u n o p r e c i p ~ o n
(with the same antibody) of translation product~ from poly
(A) RNA from placenta showed 2 major bands around 30,000
daltons. Under stringent c o n d i t i o n , the small hybridiza-
tion seen by dot-blot und~ weak conditions d~appeared.
Northern analyses of t o t a l RNA from placenta failed to de-
t e ~ mRNA of 1000 b~es s i z e l i k e in thyroid glands, but
hybridization o c c ~ e d with larger mRNA (around 4300, 2300
and 2000 b ~ ) . Dot-blot hybridization of t o t a l RNA ext~a-
~ e d from mammary ~ands were negative.
In c o n ~ i o n , o ~ r ~ u l t s s ~ g e s t that CT gene i s not
expressed i n r ~ placenta and in rat mammary gland since
CT mRNA were not d~te~ed in both t i s s u e s .

P9
Calcitonin biosynthesis in pacific salmon d u r i n g
sexual reproduction.
L. Maubras, J. Taboulet, G. M i l h a u d , M.S. M o u k h t a r a n d M..
Cressent.
U 1 1 3 I N S E R M , 2 7 R u e C h a l i g n y 7 5 0 1 2 Paris ( F R A N C E ) .
Calcitonin plasma levels show important fluctuations during the
migration of the salmon (1). We have investigated the biosynthesis of
the hormone during the sexual cycle of the pink salmon. Plasma samples
and ultimobranchial glands were obtained from male or female
pre-spawning, spawning and post-spawning fish. RNA was extracted
from the individual glands by the lithium chloride urea method. Poly A +
RNA, obtained by oligo dT chromatography was translated in an in vitro
system and the calcitonin precursor identified by immunoprecipitation
with specific antibodies for synthetic sa}mon calcitonin. Calcitonin
content of the ultimobranchial glands and plasma levels of the hormone
were measured by a specific radioimmunoassay. Important changes in
tissue and plasma levels of the hormone and it's messenger were
observed during and after spawning. These changes suggest that
calcitonin plays an important role during the sexual cycle independently
of changes in calcium content of the water.
(1) Watts, E.G., Copp, D.H. and Deftos, L.J. Endocrinology, 96, (1975)
214-218.
PI,I
Regulation of calbindin-D 28K and its mRNA in the duodenum
and uterus of the domestic hens. Nys Y*, Mayel-Afshar S.,
D.E.M. Lawson+. * INRA Nouzil]-y---~380 France, +AFRC
CambridgeCB2 4AT U. K.
Duodenal and uterine concentrations of CaBP-mRNA were
measured in immature pullets and laying hens by dot blot
analysis. In immature pullets oestrogen increased uterine
CaBPmRNA levels (control 7 + 1 ng/g - oest. 21 + 1 ng/g).
When testosterone was adm~-nistered with oest{6gens the
increase was higher in the uterus (47 + 8 ng/g) and was
also significant in the duodenum (eont~l 49 + 12 ng/g -
oest. testo 240 + 64 ng/g). In laying hens, The duodenal
concentration of--CaBP-mRNA was higher than in treated
immature pullets and was slightly modified by the formation
of the shell (respectively 411 + 70 and 556 + 89 ng/g 4 and
12 hrs after ovulation). In c[~-ntrast, the u-terine levels of
CaBP-mRNA increased markedly during formation of the shell
compared to period preceding calcification. (22 + 4 and 514
+ 77 ng/g 4 and 12 hrs after ovulation). Th-e levels of
~albindin-D 28K paralled changes in its-mRNA except during
the periods of egg formation when calbindin-D28K levels
were unchanged. The experimental suppression of shell
formation decreased levels of calbindin-D28K in both
tissues and of its mRNA in the uterus. Whereas the duodenal
levels of CaBP-mRNA was doubled in hens laying shell-less
eggs (908 + 174 ng/g). These results suggest the existence
of transla~onal or mRNA degradational control in the
duodenum. In conclusion the changes in calbindin-D 28K
levels associated with egg production are elicited by
transcriptional control in both tissues. The timing (~f the
stimulation and the levels of the increases in CaBP-mRNA
induced by sexual maturity and shell formation respectively
suggest however that the stimuli differ in the duodenum and
uterus 9
83
PXO
Identification of the precursor of a calcitonin
like p.eptide in a crustacean Nephrops
norveglcus.
Y.ARLOT-BO~INEMA~NS1, M.FOL~HERE-AU-PERON2, G.MILHAUO1, MS
MOUKHI"AR~and A. JULUENNE'-
1- UA 163 CNRS et U 113 INSERM, CHU St Antoine 27 rue Chaligny
75012 PARIS. 2 - Laboratoire de Biologie Marine, Coll4ge de France 29
110 Concarneau
We have recently shown that salmon caidtonin like molecules are
present in the plasma (1) and in the tissues of a crustacean (2).
We have studied the biesynthesis of these celcitonin like peptide by
a a crustacean tissue which contains large amounts of these molecules :
the oesophagus of Nephrops norvegicus. RNA, extracted by the phenol
chloform method, was chromatographied on oligo dl- cellulose. Poly A+
RNA obtained directed the synthesis, in an in vitro cell free system, of
multiple proteins. A large molecular weight ( M r 24000 dalton)
molecu e was specifically immunoprecipitated by antibodies raised
against synthetic saJmon calcifonin.
The successfull identification of this putative precursor of the
crustacean calcitonin like molecules opens the way to the cloning of the
respective messenger and the elucidationof its sequence.
(1). Arlot-Bonnemains, Y., Van-Wormhoudt, A., Favrel, P.,
Fouchereau-Peron, M., Moukhtar, M.S. and Milhaud G. Expedentia.
1986,42, 419-420
(2). Fouchereau-Peron M., Arlot-Bonnemains Y., MNhaud G. and
Moukhtar M.S. Gen. Comp. Endocr. 1987, 65, 179-183
P12
REGULATION OF CALCITONIN GENE TRANSCRIPTION IN VIVO BY
1,25(OH) 2D 3 . Tally Naveh-Many and Justin Silver,
Nephrology Services, Hadassah University Hospital,
Jerusalem, Israel.
We have shown that 1,25(OH) 2D 3 regulates the transcrip-
tion of the PTH gene in vivo in the rat (Silver et al.,
J Clin Invest 78:1296, 1986). For these ~xperiments rats
had been injected with 1,25(OH) 2D 3 i.p. and RNA extracts
of thyro-oarathyroid tissue were analyzed by dot blot and
blot hybridization. Because C-cells of the thyroid have
receptors for 1,25(OH) 2D (Ffeake et al., Biochem J 206:
181, 1982), we have now studied the regulation of the
calcitonin gene by 1,25(OH) 2D3 by rehybridizing the
filters used in the PTH gene regulation experiments w~th
labeled cDNA clones for calcitonin, thyroglobulin and
actin. C a l c i t o n i n m R N A levels after i00 pmol 1,25(OH) 2D 3
decreased to 6% of basal at 6 h, and 4% at 48 h. There
was no change in mRNA levels for thyroglobulin or actin.
A 1,25(OH) 2D 3 dose response curve showed a 60% reduction
in c a l c i t o n i n m R N A after 12.5 pmol 1,25(OH) 2D3, with a
maximal effect of 50 pmol. There was no increase in serum
calcium. Gel blots showed that after 1,25(OH) 2D 3 the level
of calcitonin raRNA decreased but with no change in ~ts
size. In vitro nuclear transcription showed that 1,25(0H) 2
D3 treated (i00 pmol) rats' calcitonin transcription was
5% of control, whilst thyroglobulin was 100%.
84
P13
Linkage analysis of Hypophosphataemic rickets and X-linked
DNA po!ymorphisms.
R.V.Thakker',K.E.DaviesZ,S.WoodZ,A.KingZ,B.Carrington ' ,
T.Flint 2 and J.L.H. O'Riordan'.
'The Middlesex Hospital, London WI, 2Nuffield Dept. of
Clinical Medicine, Oxford, ~Univ. of Columbia, Canada.
The X-linked hypophosphataemie rickets gene locus (HPDR)
has been localised on the short arm of the X chromos--~
and the gene order Xpter-D2-HPDR-99.6-Xcen established.
However, additional closely linked marMers are required as
frequent recombination has been observed. The locus DXSI97
has been localised to Xp21-Xpter by somatic cell hybrids
and in order to further estab'lish the map position of
DXSI97, which reveals restriction fragment length poly-
morphisms (RFLPs) at 25Kb and 20Kb for the enzyme BglII,
a linkage study in five informative Nypophosphataemic
rickets families (39 affected, 56 unaffected members) was
undertaken. X-linked RFLPs for the loci 782, D~, 99.6, C7
and 7 5 4 w e r e previously ascertained (Th~k-6r e-~ ai, J. - -
Medical Genetics, in press). Multilocus linkage analysis
was performed using the computer program LINK~IAP. Linkage
was established between DXSI97 and a group of four
X-linked loci 782-D2-HPDR-99.6; the probability of placing
DXSI97 in the most likely gene order versus a location
Uniinked to these loci being 25,000 to i. Examinabion of
multipoint crossovers revealed that the minimum number of
total recombinants was obtained by locating DXSI97 distal
to HPDR and proximal to D2. The L I N K M A P program is able
to use information from a---number of multipoint crosses to
calculate the most likely location of one unmapped gene in
a framework of well mapped markers. Within the order
Xpter-782-D2-HPDR-99.6-Xcen, the location of DXSI97 distal
~o HPDR is favoured with a probability of 130,000 to i.
Thus DXSI97 is mapped distal to HPDR and another impor-
tant. cloned DNA genetic marker for hypophosphataemic
rickets established. This will further facilitate the
constructiom of a restriction map around the disease
locus and the elucidation of the biochemical defect.
PI5
GENOMIC ORGANIZATION OF CALBINDIN-D28K (THE VITAMIN D
INDUCED CALCIUM BINDING PROTEIN)
A.W. Norman, P.P. Minghetti, L. CanceIa, Y. Fujisawa and
G. Theofan.
Division of Biomedical Sciences and Department of Bioche-
mistry University of California, Riverside CA 92521, USA.
Vitamin D through its hormonal agent 1,25-dihydroxy-
vitamin D 3 (i 25(0H) D ) is known to regulate the trans-
23
- '
erlption of many genes including the calbindins-D, para-
thyroid hormone, bone gla-protein, bone matrix protein,
x-interferon, collagen, prolactin etc. The best studied
system, in this regard, is the induction in the intestine
and kidney of a calcium-binding protein, either 28KD or i0
KD. Calbindin-D28K is known to bind 4 atoms of calcium per
mole of protein ; it is structurally related to calmodulin,
parvalbumin, troponin C and the myosin light chain
kinases. To further define the action of 1 25(0H) D upon
' 23
gene expression we have cloned and determlned the molecu-
lar organization of the chicken calbindin-D28K genome. We
have screened a l-phage chicken library by plaque hybridi-
zation using several calbindin-D28K cDNA probes. Several
overlapping clones were found to span the entire gene
which is at least 20kb in size. The gene contains many
more axons than calcium-binding domains.
Supported in part by USPHS AM 09012.23.
PI4
1,25-DIHYDROXYCHOLECALCIFEROL CONTROLS RAT VITAMIN D-
DEPENDENT CALCIUM-BINDING PROTEIN GENE EXPRESSION AT BOTH
TRANSCRIPTIONAL AND POST-TRANSCRIPTIONAL LEVELS
J.M. Dupret, P. Brun, C. Perret, N. Lomri, M. Thomasset,
p. Cuisinier-Gleizes
INSERM U.120, 78110 Le V~sinet (France).
The mechanism of action of 1,25-dihydroxycholecalciferol
(I,25(OH)zD 3) is steroid hormone-like. In mammals the in-
testinal vitamin D-dependent calci~am-binding protein is a
small 9,000 Mr protein (9K CaBP) which represents a mole-
cular marker of the hormonal action of 1,25(OH)zD 3. The in
vivo stimulation of 9K CaBP gene expression has been analy~
zed using a specific complementary DNA probe and by measu-
ring the rate of 9K CaBP gene transcription in isolated
nuclei ("run-on" assay). Our results indicate that in vi-
tamin D-deficient rats I,Z5(OH)zD 3 markedly increases 9K
CaBP gene transcription as early as 15 min after dosing,
the level of RNA synthesis reaches a maximum by lh and the
transcriptional activity decreases quite rapidly there-
after. There is an initial transient accumulation of 9K
CaBP RNA (from 7 to 15 min), which is followed by a se-
cond, significant increase, by 3h until the last time exa-
mined (iBh). In vitamin D-replete rats the hormone treat-
ment results in a small increase in 9K CaBP mRNA within
the first hour with no decline in the mRNA levels at lh.
These results could indicate the presence in duodenal
cells from vitamin D-replete animals of a stabilizing
agent which prevents transcripts from degradation. The
presence of detectable 9K CaBP premessengersonly during
the initial post injection period in vitamin D-deficient
rats may also reflect an involvement of 1,25(OH)zD 3 depen-
dent factor(s) in the mRNA processing steps.
In summary 1,25(OH)zD 3 may modulate 9K CaBP gene ex-
pression by at least two ways : a rapid transcriptional
stimulation and a potent stabilization of 9K CaBP trans-
cripts preventing their degradation and accounting for
their accumulation several hours after the hormone
treatment.
P16
LOCALIZATION OF PREPROEGF mRNA DURING
EMBRYONIC MOUSE MOLAR T O O T H DEVEL~)PMENT.
H.C. Slavkin, M.L. Snead, W. Luo a n d L.B. Rail . Lab. Dev.
Biol., School of D~ntistry, U n i v e r s i t y of S o u t h e r n C a l i f o r n i a ,
Los Angeles, a n d C h i r o n C o r p o r a t i o n , Emeryville,
C a l i f o r n i a USA.
When, where a n d h o w do e p i t h c l i a l - m e s e n c h y m a l
interactions m e d i a t e tooth morphogenesis, cytodifferentiation
a n d tissue-specific biomineralization remain challenging issues
f o r cellular, m o l e c u l a r a n d d e v e l o p m e n t a l biology. In the
mouse m o l a r tooth o r g a n model, cell p r o l i f e r a t i o n a p p e a r s to
be d o w n regulated in concert with the d e t e r m i n a t i o n of
position-restricted epithelial a n d m e s e n c h y m a l cell
polarization, a n d c h a n g e s in basement m e m b r a n e constituents
(type IV collagen, l a m i n i n , f i b r o n e e t i n , h e p a r i n s u l f a t e
p r o t e o g y l c a n ) , resulting in ameioblast a n d o d o n t o b l a s t cell
lineages. The present studies were designed to i d e n t i f y if,
w h e n a n d w h e r e p r e p r o E G F is expressed d u r i n g e m b r y o n i c
mouse m o l a r tooth d e v e l o p m e n t a n d to correlate these findings
w i t h epithelial a n d e c t o m e s e n c h y m e c y t o d i f f e r e n t i a t i o n a n d
initial b i o m i n e r a l i z a t i o n . We h a v e used sense a n d antisense
R N A probes c o r r e s p o n d i n g to the a m i n o a c i d residues 1070-
1081 of the p r e p r o E G F molecule a n d in situ h y b r i d i z a t i o n
histochemistry. Detection was by a u t o r a d i o g r a p h y f o r 5 days.
Mouse 8 week p o s t n a t a l s u b m a n d i b u l a r g l a n d was used as a
positive control. I m m u n o c y t o c h e m i c a l localization was used to
d e t e r m i n e the d i s t r i b u t i o n of basement m e m b r a n e c o n s t i t u e n t s
along the e p i t h e l i a l - m e s e n c h y m a l i n t e r f a c e . Cells of the fetal
tooth o r g a n express p r e p r o E G F d u r i n g in utero development.
These results suggest t h a t E G P per se or m o t i f ' s c o n t a i n e d
w i t h i n the p r e p r o - E G F molecule m a y serve as p a r a c r i n e
regulatory factors w h i c h control t i m i n g a n d positional aspects
of tooth d e v e l o p m e n t a n d possibly regions f o r initial
b i o m i n e r a l i z a t i o n . S u p p o r t e d by r e s e a r c h g r a n t s DE-02848,
DE-06425 a n d DE-06852, N I D R , NIH, USPHS.