changing patterns of transcript!onal and post ...genesdev.cshlp.org/content/1/10/1172.full.pdf ·...

Changing patterns of transcript!onal and post-transcriptional control of l ver-

gene expression during rat velopment

Arturo Panduro, Fouad Shalaby, and David A. Shafritz

Marion Bessin Liver Research Center, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461 USA

Genes coding for unique or tissue-specific (differentiated) functions in the liver are induced at different times during development. It has generally been felt that transcriptional control represents the dominant mechanism for regulating expression of these genes. We have determined the relative transcription rates and mRNA steady-state levels for a series of genes specifically or preferentially expressed in rat liver and find examples of transcriptional control (albumin, a-fetoprotein, a~-antitrypsin, tyrosine aminotransferase, transferrin, and cytochrome P450, TF-1) and post-transcriptional control (a~-acid glycoprotein, apolipoproteins A-1 and E, malic enzyme, and ATP citrate lyase), as well as "mixed" regulation (ligandin and cytochrome P450, RI7). Examples have been identified in which the predominant mode for regulating expression of preferentially expressed genes changes from transcriptional to post-transcriptional at different stages of liver development and some members of multigene families (cytochrome P450s and apolipoprotein genes) also show independent and sometimes contrasting modes of regulation. Therefore, it appears that regulation of specific gene expression in the liver is a dynamic process, far more complex than heretofore suspected, and a much greater contribution of post- transcriptional regulation accounts for changes in expression of genes representing major functions of the liver.

[Key Words: Liver gene expression; transcriptional and post-transcriptional control; multigene families; developmental regulation]

Received July 21, 1987; revised version accepted October 23, 1987.

Progressive changes in the production of enzymes and proteins by the liver during normal development have been known for some time. In 1977, Greengard reviewed information available in both humans and rats (Green- gard 1977) and proposed that the timing for induction of proteins unique to liver function generally occurred at three specific developmental stages: (1) late gestation, (2) at or directly following birth, and {3)just prior to weaning (---3 weeks of age in the rat). The function of genes whose expression changed at these various times was diverse and it was not clear how these genes might be related to each other or how their expression might be controlled. Other studies indicated that many physiologic and hormonal, as well as morphologic, changes occurred in the liver during development, but the molecular mechanisms responsible for these changes have also remained obscure.

In rodents, the liver develops from the gut endoderm at approximately 9 days of gestation (Thieler 1972). Be- tween days 11 and 15, the liver becomes recognizable as a physical structure, at which time it serves primarily as a hematopoietic organ (Paul et al. 1969). Certain liver- abundant genes of unknown function are already ex-

pressed at this time (Barth et al. 1982), and by day 15, c~-fetoprotein (AFP) synthesis can be detected (Abelev 1971). A process then begins in which specific genes important to liver function are expressed in a sequential fashion as this organ matures. At present, little is known about the events or factors that regulate the liver differentiation program, but this system offers an attractive opportunity to investigate specific levels of gene regulation involved in cellular and developmental stage-specific gene expression in a naturally occurring in vivo model.

Studies in rats and mice have shown that AFP mRNA has already begun to accumulate by about day 15 in utero, followed almost immediately by albumin, and Tilghman and Belayew (1982) and others (Koga et al. 1974; Sala-Trepat et al. 1979; Liao et al. 1980; Powell et al. 1984) have concluded that the level of mRNA for both of these proteins is controlled transcriptionally. Since the reduction of AFP mRNA occurs after induction of albumin mRNA, their expression is not coordi- nately regulated by a simple gene switch, even though both genes are located in tandem at a single chromo- somal locus (chromosome 5 in the mouse and chromo-

1172 GENES & DEVELOPMENT 1:1172-1182 © 1987 by Cold Spring Harbor Laboratory ISSN 0890-9369/87 $1.00

Cold Spring Harbor Laboratory Press on November 24, 2020 - Published by genesdev.cshlp.orgDownloaded from

http://genesdev.cshlp.org/

http://www.cshlpress.com

Liver gene expression during rat development

some 14 in the rat). That both genes can be active simul- taneously has been confirmed by in situ hybridization, identifying albumin and AFP mRNAs in the same cell (Poliard et al. 1986). Tilghman and Belayew (1982)proposed that induction of albumin and AFP is regulated by one set of signals and then AFP is shut off by a separate signal, while albumin expression continues. These signals are controlled by separate genetic loci, two of which have been identified, rif and raf (Belayew and Tilghman 1982).

Both transcriptional and post-transcriptional steps are essential in controlling expression of a given gene in a specific tissue under a given set of circumstances (Dar- nell 1982). With the availability of many probes, we have instituted an analysis of the relative contribution of transcriptional and post-transcriptional events in the regulation of specific genes in rat liver during development. Three categories of genes have been analyzed; (1) those expressed uniquely in liver (liver-specific genes), (2) those expressed preferentially in liver and one or a few other cell types (liver preferentially expressed genes) and (3)those expressed in all cells (general cellular genes). Powell et al. (1984)previously examined a series of such genes in the mouse and concluded that differential transcription appeared to be the basis for the controlled time of appearance of liver-specific mRNAs during development, although instances of post-transcriptional control were also apparent. Our present studies in rats, using a larger panel of genes of known function, indicate substantial variability in the dominant mode of regulation for genes expressed in liver, independent of whether these genes serve liver-specific or more general functions. For several genes, we have found that the level of regulation, i.e., transcriptional or post-transcriptional, changes during development. Inde- pendent members of multigene families also show independent and sometimes contrasting modes of regulation.

Results

To standardize conditions under which expression of specific genes was measured, pregnant rats and groups of offspring born on the same day were placed in the same conditions of environment and alimentation. Total body weight was determined periodically until the rats were 18 weeks old. During this period, rats of the same group were killed at different times to determine liver wet weight. Three different stages of development were studied: (1)late gestation, (2)the first 6 weeks post partum, and (3)maturat ion between ages 6 and 18 weeks. The period of most rapid rat growth occurred between ages 2 and 10 weeks (Fig. 1B). However, the period of most rapid liver growth occurred between ages 2 and 6 weeks (Fig. 1A). In addition, during the first 6 weeks of life, there was a greater rate of increase in liver weight compared to total body weight (Fig. 1C).

Transcriptional regulation of liver-specific and preferentially expressed genes

In previous studies, we observed changes in the relative

"r

0

n

-5

A v

I - . " r '

(.9

I.iJ

f i r 1.1.1 >

_1

20"

I0 -

5 0 0 -

"" 4 0 0 - I....- "r"

o 300- I.iJ

200- ),.. Q 0

I00-

A

1 I I I I I I I I

B

I , / I

/ , / I 1 I I

o.o5~ C / . ~

. ::1 / I"-~.1~ ~ ' .

• 002_[,/" o

I I ! I

~ °

AGE OF RATS (weeks)

Figure 1. Growth pattern of rat liver compared to total body weight.

transcription rate of the albumin gene associated with normal rat growth (Panduro et al. 1987). We extended these studies to different times during development and found that albumin transcription, which was already measurable at day 17 in utero, increased to a maximum at 2 -3 weeks after birth and then decreased (Fig. 2). On the 17th day in utero, transcription of the AFP gene was higher than albumin; it then decreased after birth and became undetectable by ages 4 -6 weeks.

To determine whether the levels of specific mRNAs corresponded to the relative transcription rates for these genes, total RNA was isolated from separate portions of the liver and the steady-state level of specific mRNA determined by Northern blot hybridization (Fig. 2). For both albumin and AFP, the steady-state level of mRNA in total hepatic tissue increased and decreased in con- junction with the relative transcription rate.

Tyrosine aminotransferase (TAT) is another gene whose expression represents a unique function of the liver (Scherer et al. 1982). Minimal transcriptional activity was detected before or at the time of birth (Fig. 3), after which transcription increased dramatically in the first 4 weeks and then remained essentially constant

GENES & DEVELOPMENT 1173




Panduro et al.

Figure 2. Relative transcription rates and mRNA steady-state levels of albumin and AFP genes during development of rat liver. Nascent RNA molecules in the process of transcription were labeled in isolated nuclei with [32p]UTP. 10 x 106 cpm of labeled RNA were then hybridized to cloned specific cDNA sequences fixed in large excess to individual nitrocellulose filter discs. Hybridized labeled RNA was then determined for each disc, as noted in Materials and methods, with subtraction of background values of 20-25 cpm, obtained with nonrecombinant plasmid pBR322. Results represent average values for three separate experiments. Bars indicate standard deviations for individual points. (N.B.) Newborn animals. For Northern blots, 10 ~g of total RNA extracted from a separate portion of the liver used for in vitro nuclear RNA labeling, was applied to each lane of a 1% agarose gel under denaturing conditions, and electrophoresed and handled subse- quently as noted in Materials and methods. In the series of RNA extracts used for detecting albumin sequences, the sample used for day 19 in utero had an inordinately low signal, which was found for all mRNAs tested in this sample.

z ~ 30 l

o o

-E rr

z ,~ 1.0

~ z

AIb.

AFP.

AIb

B AFP

~ NB I 2 4 6 9

DAYS IN AGE OF UTERO RATS (Weeks)

14

17 19N.B. I 2 4 6 9 14 18

t "tlillil/ m

8j

Z oa--

K-"

Z u <1: ""

0.2 . J

z r ~ z z a

I"- -0.1 ~ _ j

o

IJ.

into adulthood. Except for the 2-week sample, which probably represented a technical problem in mRNA ex- traction, the steady-state level of TAT mRNA correlated with the relative transcription rate of this gene.

Transferrin is an iron binding protein synthesized principally in the liver but is also produced in other cells, such as oviduct, testes, brain, and bone marrow (Idzeda et al. 1986). Transcription of the transferrin gene (Fig. 4)was initiated in utero, reached a peak when the rat was 1 week old, decreased during the period of rapid liver growth (age 2 -6 weeks), and then became essentially constant. The steady-state level of transferrin mRNA again correlated with the relative transcription rate. Therefore, although the time of maximal transcriptional activity (and also probably initial induction) varied during rat development for AFP, albumin, TAT, and transferrin, all four genes showed a predominant transcriptional influence in determining the steady-state level of their respective mRNAs.

Post-transcriptional regulation of liver-specific and preferentially expressed genes

c~l-Acid glycoprotein (pAGP) is a so-called "acute-phase" reactant found in the serum (Ricca et al. 1981). It is one of several plasma proteins synthesized specifically by the liver in response to stressful stimuli including sur- gical trauma, bacterial infection, and nonspecific inflammation. Transcriptional induction of pAGP was noted at birth (Fig. 5A); it then decreased gradually over the next 2 weeks and became unmeasurable thereafter (a cpm level twice that obtained with nonrecombinant plasmid pBR322 was considered necessary before results

were interpreted as positive for any given gene), pAGP is glucocorticoid regulated (Baumann et al. 1983; Vannice et al. 1984)and its expression at birth could be related either to the traumatic process of delivery or to a surge in steroids in the mother during parturition.

A high level of pAGP mRNA was also found at birth (Fig. 5A), but it was not present in the liver of 1- to 2- week-old rats. At approximately age 4 weeks, pAGP mRNA reappeared and remained elevated, even though

A

z b - - ~ 0.4 z

n _ 0.3 u

m ~ 0.2 z , ~ z ~ o I..-w ,~,~ 0.1 z j

m_

TAT 3

n n N N.B

DAYS IN UTERO

17 19 N.B.

O TAT

I I

I

L I 2 4 6 9 la j

AGE OF RATS (Weeks)

I 2 4 6 9 14 18

Figure 3. Relative transcription rates and mRNA steady-state levels for the tyrosine aminotransferase (TATa) gene during rat liver development.

1174 GENES & DEVELOPMENT





- t z o - "; 0.4 Tronsferrin Z

Q ._ 0.3

0.2 z

k-

=g, _m

N.B t l 2 4 6 9 14 I


df "m '~ - : - 0"- ~ -~

17 19 N.B. I 2 4 6 9 14 18

Tronsferrin N

Figure 4. Relative transcription rates and mRNA steady state- levels for the transferrin gene during rat liver development.

specific transcription of the gene was not detected at this time. These unexpected findings were reproduced in three separate series of experiments. The same RNA preparations were also used successfully for detection of malic enzyme and ATP citrate lyase mRNAs at 1 -2 weeks (Fig. 5B), indicating that the mRNA was not non- specifically degraded. These experiments suggest that once the pAGP gene is induced during development, its mRNA is regulated by a post-transcriptional mechanism.

Malic enzyme (ME)and adenosine triphosphate citrate lyase (ATPcl)are highly regulatable enzymes involved in fatty acid synthesis (Sul et al. 1983, 1984). They are more abundant in liver than in other tissues. Both are induced in the perinatal period and are hormonally, as well as nutritionally, regulated (Sul et al. 1983, 1984). Relative transcription rates for ME and ATPcl were low {two- to three-fold above background) but appeared to be induced in the perinatal period, decreasing to undetectable levels in growing and adult rats (Fig. 5B). The steady-state level of mRNAs for ME and ATPcl increased dramatically at birth and essentially remained constant until adulthood, even though their transcription rates decreased to essentially undetectable levels. This suggests that a post-transcriptional mechanism is also the primary means for regulating the mRNA steady-state levels for malic enzyme and ATPcl.

Transcriptional versus mixed regulation of liver preferentially expressed genes

cxl-Antitrypsin (~-AT) and ligandin are genes of major importance in liver function, although both are expressed in other differentiated cell types but not in all cells (Mannervik 1985; Kelsey et al. 1987). Transcription of c~-AT was relatively high in utero, increasing mod- estly in the first 4 weeks of life and then decreasing to an adult level that was slightly lower than that in utero

(Fig. 6). The high level of cxl-AT gene transcription in the perinatal period was associated with initial appearance of the mRNA. The steady-state level of c,I-AT mRNA appeared to correlate with the transcription rate of the gene (Fig. 6).

Transcription of ligandin was not detected in utero or in newborn rats; it increased dramatically in the first 2

z_O X

o.,o- V-.N

z

~: a 0.05 i.- LIJ

z _m

A ~J Oi- ocid glycoprotein

(pAGP)

pAGP

17 19 NB I 2 4 6 9 14 • y..__J k -~r / DAYS IN AGE OF UTERO RATS (weeks)

17 19 N.B. I 2 4 6 9 14 18

i z g

c) 0.I0- i--

~,- o 0.05- i-- i,i

=g , car)

B BE Malic Enzyme (M.E.)

[ 7 ATP citrate lyon (ATPcl)

17 19 NB I 2 4 6 9 14 __J

DAYS IN AGE OF UTERO RATS (weeks)

17 19 N.B. I 2 4 6 9 14 18

. . ( . I t

ATPct

Figure 5. Relative transcription rates and mRNA steady state- levels for pAGP, malic enzyme, and ATP citrate lyase genes during rat liver development. (AI pAGP, (BI ME and ATPcl.





Panduro et al.

wl

z9 -- =c z o EO.2

a.__ o

z < 7 ~o 0.I I,- la~J

z

(:Z=-AT

D Oi-Antitrypsin

B Ligondin

191 NB. I I 2 4 6 9 " 14 J DAYS IN AGE OF

U T E ~ RATS ( ~ $ )

17 19 N.B. I 2 4 6 9 14 18

o

Ligandin , ~

Figure 6. Relative transcription rates and mRNA steady-state levels of al-AT and ligandin genes during development of rat liver.

weeks after birth and then decreased to undetectable levels in postweanling and adult rats (Fig. 6). The initial increase in ligandin transcription after birth was associated with an increase in the steady-state level of ligandin mRNA. However, although transcription of the ligandin gene could not be detected at later ages, the steady-state level of ligandin mRNA showed a secondary rise and remained high into adulthood (Fig. 6). This suggested a change from transcriptional to post-transcriptional (i.e., mixed)regulation in controlling the level of ligandin mRNA during rat development.

In the above experiments, the relative transcription rate for o~I-AT was higher than ligandin during all stages of development (Fig. 6). However, in Northern blots using the same amounts of total RNA and cDNA probes of approximately the same length and specific activity, there was much greater hybridization with ligandin than with al-AT. This indicates that ~]-AT transcripts are turning over much faster than ligandin transcripts. In addition, a change in mRNA processing or half-life appears to be associated with accumulation of ligandin mRNA in the adult compared to the weanling rat.

Differential expression of genes within a given multigene family

The apolipoprotein and cytochrome P450 genes are highly expressed in liver b u t a r e also expressed to varying degrees in several other tissues (Eisenberg 1984; Lou et al. 1986; Achison and Adesnik 1983; Omiecinski 1986). When we previously studied expression of cytochrome P450 genes during liver regeneration (Panduro et al. 1986), we observed that two members of this family (R-17, phenobarbital inducible; and TF-1, constitutive) are regulated independently. To compare these

1176 GENES & D E V E L O P M E N T

results with events occurring during normal liver cell growth, we analyzed the transcription and mRNA steady-state levels of the apolipoproteins (Apo)A-1 and E and cytochromes P450, R-17, and TF-1 at different stages of development. Both cytochrome P450 genes were transcriptionally active at very low levels in utero and were induced shortly after birth (Fig. 7A). R-17 transcription then decreased to adult levels, whereas TF-1 transcription continued to increase up to about 8-10 weeks of age and then leveled off. The steady-state level of R- 17 mRNA showed a biphasic pattern, with one peak

A P-O ¢---- O. E

Eel I , -J

_ 0.1

g t 17 19j N.B. L I

DAYS IN UTERO

17 19 N.B. I

B R-17 D TF-I

2 4 6 9 I e j AGE~oF

RATS (Weeks) . . . . . . .

2 4 6 9 14 18"

T F - I

B ~ ' 0 ApoE

Zb-- I Apo A-I . - ,, 0.5 0 ~- ~ 0.4 n _

u 0.3

< 0.2 n,"O I,.-. W

<~moZ'J~ 0.i

N.B. t l 2 4_ 6 ~ 9 14 18j


F

17 19 N.B. I 2 4 6 9 14 18

ap0E • lit

,oo,-, | g e O O - . , - "

Figure 7. Relative transcription rates and mRNA steady-state levels for cytochromes P450, R17, and TF-1 and Apo A-1 and E genes during rat liver development. (A) Cytochromes P450, R-17, and TF-1. (B) Apo A-1 and Apo E.





before birth and a second peak in the postweanling rat. Before birth, changes in the mRNA level for R-17 correlated with transcription; however, after birth, R-17 mRNA accumulated when transcription was decreasing (Fig. 7A). This represents transcriptional control of R-17 in utero, but post-transcriptional control in the growing rat.

In contrast, TF-1 mRNA was not detected in utero, although there was a very low transcriptional signal at this time. Essentially, the TF-1 gene turned on after birth and the mRNA steady-state level increased in con- junction with the relative transcription rate. Therefore, these two related cytochrome P450 genes not only show different patterns of expression during development and are regulated independently, but also use different mechanisms to control their expression, i.e., transcriptional for TF-1 and mixed for R-17.

Expression of Apo A-1 and Apo E genes was also regulated independently (Fig. 7B). Transcription of Apo E was high in utero; it decreased at birth, increased and decreased again, and then approached adult levels. Tran- scription of Apo A-1 was active in utero; it went up and down during rat growth and then returned to higher levels in adult rats. The most important observation concerning expression of Apo A-1 and Apo E was the discrepancy in both cases between gene transcriptional activity and the mRNA steady-state level. With Apo E, transcription was high in utero but the mRNA level was low. This probably means that Apo E mRNA is turning over very fast in utero; either it has a short half-life or a large portion is degraded in the nucleus without ever en- tering the cytoplasm. After birth, Apo E mRNA accu- muated to high levels, but the transcription rate was about the same as in utero. Therefore, it would seem that the mRNA half-life changed after birth or more was released into the cytoplasm. In either case, this would suggest a change in the post-transcriptional mechanism governing the level of Apo E mRNA during development.

Transcription of the Apo A-1 gene was much lower in utero than Apo E (Fig. 7B). However, the Apo A-1 mRNA steady-state level in utero was relatively high, illus- trating the importance of post-transcriptional control in regulating expression of this gene. When transcription of Apo A-1 increased after birth (1-2 weeks), the mRNA steady-state level also increased. However, when transcription decreased at 4 - 6 weeks to levels comparable to those observed in utero (Fig. 7B), there was a much greater proportional decrease in the steady-state level of Apo A-1 mRNA, implying either a shortening of the Apo A-1 mRNA half-life or a reduction in the amount of Apo A-1 mRNA released from the nucleus. This would suggest a negative form of post-transcriptional control in regulating the level of Apo A-1 mRNA. Thus, the mRNA steady-state level for two related apolipoprotein genes (Apo A-1 and Apo E) appears to be regulated by post-transcriptional changes in apparent mRNA stability or half-life. However, these changes occur in opposite directions, leading to a high steady-state level of Apo E mRNA and a low steady-state level of Apo A-1 mRNA in post-weanling and adult rats.

Expression of general cellular genes during rat liver development

Transcription of the general cellular genes, ~-actin and ~-tubulin showed two peaks of transcriptional activity, one in utero and the other shortly after birth (Fig. 8). The steady-state levels of fPactin and ~-tubulin mRNAs were highest in utero, much higher in proportion to their relative transcription rates than after birth, again suggesting post-transcriptional regulation. With the 13- actin probe, there were two RNA transcripts detected at steady state. The upper transcript, which is slightly larger than 18S ribosomal RNA, probably represents smooth muscle actin, whereas the lower transcript represents ~3-actin with a small contribution of 7-actin, which is approximately the same size (L. Kedes, pers. comm.). The upper actin RNA band did not show a secondary rise between 1 and 4 weeks after birth, whereas the lower band (f~-actin)showed this rise, corresponding with increased transcription.

With cx-tubulin, there was no significant rise in the mRNA steady-state level accompanying the secondary increase in transcription of this gene after birth {Fig. 8). These findings are consistent with previous studies of Powell et al. (1984), in which the relative abundance of tubulin mRNA compared with its relative transcription rate was higher in fetal as compared to adult liver.

D i s c u s s i o n

In attaining a final pattern of differential expression of specific genes in the liver, there are at least three major phases in development that can be readily defined: com- mitment of embryonal cells to become hepatocytes,

i z -° 0.3

Q._ ~:w 0.2 U J

Z

n-

• t ~ o., Z

/ ~ - A c t i n

t 17 19 j N.B. 1

DAYS IN UTERO

. .r

17 19 N.B. /

B-Actin

~ Q.Tul~lln

I 2 4 6 9 14~

AGEOF RATS (Weeks)

2 4 6 9 14 18

a -Tubulin

Figure 8. Relative transcription rates and mRNA steady-state levels for 13-actin and a-tubulin genes during rat liver development.

GENES & D E V E L O P M E N T 1177




Panduro et al.

modification in expression of specific genes during late fetal life in preparation for extrauterine function, and maturation of the newborn liver to assume an adult pat ° tern of differentiated function. During each of these pe- riods, there are significant anatomic, morphologic, and physiologic changes that occur and these changes un- doubtedly play a significant role in regulating expression of specific genes in this organ.

To begin to understand the factors that might con- tribute to the liver developmental program, a number of laboratories have begun to use molecular probes to study the patterns of expression of specific genes under various circumstances, e.g., during normal development (Sala- Trepat et al. 1979; Liao et al. 1980; Tilghman and Be- layew 1982; Meeham et al. 1984; Powell et al. 1984; Sellem et al. 1984; Giachielli and Omiecinski 1986; Po- liard et al. 1986), during liver regeneraton (Friedman et al. 1984; Tamaoki and Fausto 1984; Panduro et al. 1986), in different regions of the hepatic lobule (Chianale et al. 1986), in isolated hepatocytes (Clayton and Darnell 1983; Jefferson et al. 1984; Jefferson et al. 1985), and in various hepatoma cell lines, variants, and somatic cell hybrids (Peterson and Weiss 1972; Darlington et al. 1974; Peterson 1976; Clayton et al. 1985b). Most of these studies have been limited to analysis of steady-state levels of one or a few related mRNAs under a variety of circumstances. However, Darnell and co-workers (Derman et al. 1982; Clayton and Darnell 1983; Friedman et al. 1984; Jefferson et al. 1984; Powell et al. 1984; Clayton et al. 1985a, 1985b) have performed more extensive analysis by measuring both the relative transcription rates and specific mRNA levels for a series of genes unique or differentially expressed in liver compared with genes commonly expressed in all higher eukaryotic cells. Their original findings (Friedman et al. 1984; Powell et al. 1984)suggested that transcriptional control represented the primary mechanism regulating the level of mRNAs for liver-specific functions. How- ever, more recent in vitro studies by these investigators using tissue slices, isolated hepatocytes, and hepatoma cell lines (Clayton and Darnell 1983; Jefferson et al. 1984; Clayton et al. 1985a, b)suggest an additional contribution of post-transcriptional regulation. In addition, cell-cell contact appears to play a major role in liver- specific gene expression at the level of transcription (Clayton and Darnell 1983; Clayton et al. 1985a).

The present study represents an extension of this analysis in the naturally occurring in vivo system of liver development, using a variety of genes representing major known hepatic functions. When this study was started several years ago, we were hoping to find a simple cate- gorization of genes of known function that would be regulated by fixed mechanisms initiated at specific times. However, our results (summarized in Table 1) are far more complicated than originally anticipated. Genes have been divided into three classes: liver-specific (albumin, AFP, TAT, and pAGP), liver preferentially expressed (c,I-AT; ligandin; transferrin; Apo A-1 and Apo E; cytochrome P450s, R-17, and TF-1; malic enzyme; and ATP citrate lyase), and general cellular genes ([3-

Table 1. Mechanisms controlling the steady-state level of specific mRNAs during development of rat liver

Post- Gene Transcrip- transcrip- category tional tional "Mixed"

Liver-specific Alb, AFP, pAGP TAT

Preferentially ~t-AT, M.E., ATP-cl, ligandin, expressed transferrin, Apo A- 1, cytochrome

cytochrome Apo E P450, R17 P450, TF- 1

General [3-actin [3-actin cellular o~-tubulin

actin and a-tubulin). Examples of specific genes regulated primarily at the transcriptional or post-transcriptional level have been found in each of these categories. Post-transcriptional regulation has been reported previously as a commonly used mechanism for house- keeping genes, such as actin and tubulin (Friedman et al. 1984; Powell et al. 1984), thymidine kinase (Groudine and Casimir 1984), and dihydrofolate reductase (Leys et al. 1984), as well as cell-cycle-dependent nuclear protein genes, e.g., myc (Marcu 1987) and certain histone genes (Schumperli 1986).

Several liver-specific or preferentially expressed genes also exhibited complex mechanisms of control. This was independent of whether these genes were induced in late gestation or following birth. Not only were different liver-specific and preferentially expressed genes induced at different times during development, but the predominant mode for regulating specific genes (transcriptional or post-transcriptional) could vary at different times. Ex- amples of genes showing such mixed regulation include ligandin, cytochrome P450, R-17, and possibly [3-actin (Table 1 ).

A surprising finding was that different members of multigene families (cytochrome P450s and apolipoproteins) showed different and varying modes of regulation during development. For Apo A-1 and Apo E, changes in apparent mRNA stability occurred in opposite directions, indicating both positive and negative elements of post-transcriptional control. Aside from Apo A-I, the only other mRNAs showing an apparent decrease in stability after birth were [3-actin and c~-tubulin. AFP expression was also reduced (virtually absent)after the first few weeks of life, but in this case transcription of the gene also became unmeasurable.

In recent studies in rat hepatoma cell line variants and revertants, Clayton et al. (1985b)have shown that a post-transcriptional mechanism, termed mRNA conservation, leads to increased steady-state levels of liver-specific mRNAs when their transcription rates are low. In the present study, mRNA conservation appears to represent the predominant mechanism for accumulating malic enzyme, ATP citrate lyase, and pAGP mRNAs in the postnatal period. It probably also contributes to the relatively high steady-state levels of ligandin and Apo E mRNA after birth. Whether mRNA conservation results






from increased processing of primary transcripts, more efficient nucleo-cytoplasmic transfer of mRNA, increased mRNA stability, or a combination of these factors remains to be determined.

At present, the specific factors responsible for different modes of regulation for individual genes during liver development are not known. After its initial induction in late gestation, albumin transcription goes through a sharp developmental peak after birth, during the period of rapid liver growth (age 2 - 6 weeks). At this time, there is morphological reorganization of hepatocytes within the liver lobule to increase cell-cell contact. As indicated above, Clayton and Darnell (1983)and Clayton et al. (1985a), have recently demonstrated that such contact is essential in maintaining transcription of liver-specific genes at high levels. Reid and co-workers (Jefferson et al. 1984, 1985; Reid et al. 1986) have also shown the importance of cell-surface and extracellular matr ix components, as well as hormones, in maintaining expression of liver-specific genes in primary hepatocyte cultures.

Other factors that are known to stimulate albumin expression in vivo are insulin, glucagon, possibly other hormones, and nutritional factors (Jefferson et al. 1983; Yap et al. 1978). Insulin production also increases mark- edly in the first few weeks of life (Kakita 1983). Hor- monal signals have clearly been shown to alter the half- life of certain eukaryotic mRNAs, e.g., casein (Guyette et al. 1979)and vitellogenin (Brock and Shapiro 1983), and eukaryotic mRNAs known to be glucocorticoid regulated include growth hormone (Diamond and Goodman 1985), tyrosine amino transferase (Scherer et al. 1892), tryptophan oxygenase (Danesch et al. 1982) ~u-globulin (Kurtz et al. 1976; Fulton and Birnie 1985), and pAGP (Baumann et al. 1983; Vannice et al. 1984). In the latter two instances, improved processing or nucleo- cytoplasmic transport of the mRNA has been suggested (Kurtz et al. 1976; Vannice et al. 1984).

Although there is probably a small increase in the number of hepatocytes in the liver after birth (Le Bouton 1974), the major change is an increase in the mass of hepatocytes and morphological differentiation at both the cellular and organ levels. Both the alb/AFP gene switch and changes in expression pattern of cytochrome P450 genes during development are similar to changes we have previously identified during liver regeneration (Panduro et al. 1986). This holds true with respect to both the timing of the switch and reexpression of these specialized genes. For the albumin/AFP gene switch, this is readily apparent. For cytochrome P450 genes, in development, R-17 transcription turns on after birth, de- creases from 2 to 9 weeks, and then rises to adult levels. TF-1 is also induced shortly after birth, but its relative transcription rate then increases by a factor of threefold, peaking between 6 and 9 weeks and falling back to adult levels. In liver regeneration (Panduro et al. 1986), transcription of R-17 shows little change (a small drop on day 2 with a rapid return to normal). However, TF-1 shows a marked drop on day 1 followed by a return to normal on day 2 and then a rise to two to three times

normal on day 7. This pattern during regeneration is consistent with the late induction of TF-1 during rat development (Fig. 7.).

Conclusion

With increasing growth and specialization of function in the hepatocyte, which occurs in the first few weeks after birth, we have noted extensive changes in expression of specific genes. For example, albumin transcription and mRNA steady-state levels increase, whereas AFP de- creases. TAT, transferrin, and ligandin are induced, as well as cytochromes P450, R-17, and TF-1, but the mechanisms regulating expression of these latter two genes are separate and distinct, even though they are members of a multigene family. Apo A-1 and Apo E, members of another multigene family, also show independent and contrasting modes of post-transcriptional regulation. Some of these genes are regulated transcriptionally (alb, AFP, TAT, ~I-AT, transferrin, and cytochrome P450, TF-1), whereas others are regulated post-transcriptionally (pAGP, M.E., ATPcl, Apo A-I, and Apo E ) o r by mixed mechanisms (ligandin and cytochrome P450, R-17).

Finally, it should be noted that the tissue specificity in expression of individual genes and possibly the primary mode for regulation of a specific gene in a given tissue may vary in different species. Kelsey et al. (1987)have recently reported that there are differences in the relative expression of ~ -AT in different tissues in humans versus mice, and that these differences are preserved when the human ~-AT gene is introduced transgeni- cally into the mouse. The distinction between tissue- specific versus preferential expression of individual genes is also becoming less clear, as more sensitive methods of mRNA detection are revealing low levels of transcripts in tissues other than those in which tissue- specific expression has been reported. The present study illustrates that the level, extent, and mechanisms controlling specific gene expression are multifactorial and highly variable for individual genes during normal development of rat liver and point to the need for additional studies to elucidate the mechanisms underlying this developmental regulation.

Materials and methods

Animals

Liver and total body weight of newborn Sprague-Dawley rats (Marland farms) was determined during the growth of the animals until 18 weeks of age. Animals were housed in 21°C with 12-hr dark-light cycles and were fed Purina rat chow and water ad libitum. The timing of impregnation of the rats was indicated by the supplier. At the times indicated in various figures, rats were killed by vertebral dislocation and the liver removed quickly and perfused with a solution of 0.25 M sucrose, 1 mM MgC12 and 10 U/ml of heparin, except for newborns and fe- tuses, which were not perfused.

Isolation of Nuclei

The liver was minced and homogenized at 4°C in 5-10





Panduro et al.

volumes of 0.3 M sucrose, 10 mM Tris HC1 {pH 7.5), 5 m ~ MgC12, 5 mM dithiothreitol, and 0.1% Triton X-405. The homogenate was filtered through four layers of cheesecloth and centrifuged at 800g for 5 rain at 4°C. The pellet was resuspended in homogenizing buffer without Triton X-405, and pure nuclei were obtained by sedimentation through a sucrose gradient, according to the method of Lamers et al. (1982), as previously described (Panduro et al. 1986).

RNA transcription

In vitro labeling of nuclei was performed as described previously {Panduro et al. 1987). A total of 2.5-4.0 x 107 nuclei were incubated for 25 min at room temperature in a 250-~1 reaction mixture containing 25% glycerol, 75 mM HEPES (pH 7.5), 5 mM MgClz, 100 mM KC1, 4 mM dithiothreitol, 0.5 mM GTP, 0.5 mM cytosine triphosphate, 1.0 mM ATP, and 50 ~Ci of [et-a2P]UTP (sp. act. 410 Ci/mmole, Amersham/Searle Corp). The reaction was terminated by addition of deoxyribonuclease I (DNase I) to a final concentration of 20 ~g/ml and further incu- bation for 5 rain. Labeled RNA was isolated by the method of Groudine et al. { 1981 ).

Binding of cloned cDNAs to nitrocellulose filters

Cloned specific cDNAs were denatured in 0.1 M NaOH. Sixteen volumes of 2 m NaC1 were then added and DNA was bound to individual nitrocellulose filter disks Grade BA 85, 0.45-~m pore size, 25-ram diameter (Schleicher & Schuell), essentially as described by Gillespie and Spiegelman (1965). The following cloned cDNA probes, kindly provided by the individuals noted, were used: albumin (D. Shafritz), AFP {T. Sargent), ~I-AT (K. Krauter), TAT (G. Schutz), transferrin (J. Griswold), ligandin and pAGP (J. Taylor), Apo A-1 and Apo E (L. Chan), cytochrome P450s, RF-1 (phenobarbital inducible, subfamily 2B)and TF-1 (constitutive, subfamily 2C)(M. Adesnik), malic enzyme and ATP citrate lyase (C. Rubin), and [3-actin and a-tubulin (D. Cleveland). Except for [3-actin and a-tubulin, which were de- rived from chicken, all probes were of rat origin.

Assay for specific gene transcription products

Determination of the percent transcription of specific gene products was performed by filter hybridization as described previously (Panduro et al. 1986), using liquid scintillation spectros- copy of individual filter discs to quantitate results. Similar results were obtained using a series of cloned DNAs applied as dots to a nitrocellulose filter sheet, followed by hybridization with labeled RNA, autoradiography, and densitometry scan- ning.

Isolation of total cellular RNA

RNA was isolated using minor modifications of the procedure of Chirgwin et al. (1979). A portion of the liver was dropped into liquid nitrogen, pulverized, and then homogenized in 3.5 ml of 4 M guanidine thiocyanate solution, using a Polytron homoge- nizer. The homogenate was cleared of cellular debris by centrif- ugation at 5000 rpm for 10 min at 10°C in an HB-4 rotor and the RNA pelleted through a CsC1 gradient. The resultant RNA was redissolved in 10 mM Tris HC1 {pH 7.4), 1 mm EDTA, adjusted to 0.1 M Na acetate (pH 5.5), reprecipitated with 2.5 volumes absolute ethanol at - 20°C, resuspended in 10 mM Tris HC1 (pH 7.4), 1 mM EDTA, quantitated by A26 o spectrophotometry, and used for molecular hybridization.

Hybridization analysis of total RNA transferred to a membrane filter

Ten micrograms of total RNA was denatured for 15 min at 60°C in buffer containing 50% deionized formamide, 6% formalde- hyde, and 1 x MOPS buffer (20 mM morpholinopropanesulfonic acid, 5 mM sodium acetate, and 1 mM Na2 EDTA), placed in separate lanes of a 1% agarose gel prepared in 1 x MOPS buffer with 6% formalydehyde, and electrophoresed for 4 -5 hr at 70 mA, essentially according to the method of Thomas (1980). After electrophoresis, the RNA was transferred to a GeneScreen filter sheet (New England Nuclear Corp., Boston, Massachu- setts), as described by the manufacturer. The amount of RNA in each lane of the gel was judged to be constant by ethidium bro- mide fluorescence, identifying specific bands of 18S and 28S RNA directly in the gel and after transfer of the RNA to the membrane filter. Although it would be helpful to have an additional standard for mRNA, none so far has been demonstrated to be constant during liver development. Cloned cDNAs were labeled radioactively to a specific activity of 6 x 108 cpm/~g by the method of primer extension (Summers 1975), using [a2p] dCTP (sp. act. 3000 Ci/mmole), and were used for hybridization under stringent conditions as described previously {Panduro et al. 1986). After hybridization, filters were washed and exposed to autoradiography at -86°C on Kodak XAR-5 film, using Du- pont Lightening Plus intensifier screens.

A c k n o w l e d g m e n t s

The authors thank Ms. Ethel Hurston for excellent technical assistance, Mr. Roy Forbes for typing the manuscript, and the various investigators cited for kindly providing or permitting use of specific cDNA clones for these studies. This research was supported in part by National Institutes of Health grants DK- 17609 and DK-P30-17702.

R e f e r e n c e s

Abelev, G.J. 1971. Alpha-fetoprotein in ontogenesis and its as- sociation with malignant tumors. Adv. Cancer Res. 14: 295-358.

Achison, M. and M. Adesnik. 1983. A cytochrome P-450 multigene family: Characterization of a gene activated by phenobarbital administration. ]. Biol. Chem. 258:11285-11295.

Barth, R.K., K.W. Gross, L.C. Gremke, and N.D. Hastie. 1982. Developmentally regulated mRNAs in mouse liver. Proc. Natl. Acad. Sci. 79: 500-504.

Baumann, H., G.L. Firestone, T.L. Burgess, K.W. Gross, K.R. Yamamoto, W.A. Held. 1983. Dexamethasone regulation of c~x-acid glycoprotein and other acute phase reactants in rat liver and hepatoma cells. I. Biol. Chem. 258: 563-570.

Belayew, A. and S.M. Tilghman. 1982. Genetic analysis of e~- fetoprotein synthesis in mice. Mol. Cell. Biol. 2: 1427- 1435.

Brock, M.L. and D.J. Shapiro. 1983. Estrogen stabilizes vitellogenin mRNA against cytoplasmic degradation. Cell 34: 207-214.

Chianale, J., C. Dvorak, M. May, and J.I. Gumucio. 1986. Heter- ogeneous expression of phenobarbital-inducible cytochrome P-450 genes within the hepatic acinus in the rat. Hepatology 6:945-951.

Chirgwin, J.M., A.E. Przbyla, R.J. MacDonald, and W.J. Rutter. 1979. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18: 5294- 5299.

Clayton, D.E. and J.E. Damell, Jr. 1983. Changes in liver-spe-






cific compared to common gene transcription during primary culture of mouse hepatocytes. Mol. Cell. Biol. 3: 1552-1561.

Clayton, D.F., A.L. Harrelson, and J.E. Damell, Jr. 1985a. De- pendence of liver-specific transcription on tissue organiza- tion. Mol. Cell. Biol. 5: 2623-2632.

Clayton, D.F., M. Weiss, and J.E. Damell, Jr. 1985b. Liver-specific RNA metabolism in hepatoma cells: Variations in transcription rates and mRNA levels. Mol. Cell. Biol. 5: 2633- 2641.

Danesch, V., S. Hashimoto, R. Renkawitz, and G. Schutz. 1983. Transcriptional regulation of the tryptophan oxygenase gene in rat liver by glucocorticoids. J. Biol. Chem. 258: 4750- 4753.

Darlington, G.J., H.P. Bernhard, and F.H. Ruddle. 1974. Human serum albumin phenotype activation in mouse hepatoma- human leukocyte cell hybrids. Science 185: 859-862.

Darnell, J.E. Jr. 1982. Variety in the level of gene control in eukaryotic cells. Nature 297: 365-371.

Derman, E., K. Krauter, L. Walling, C. Weinberger, M. Ray, J.E. Damell, Jr. 1982. Transcriptional control in the production of liver-specific mRNAs. Cell 23:731-739.

Diamond, D.J. and H.M. Goodman. 1985. Regulation of growth hormone messenger RNA synthesis by dexamethasone and triiodothyronine transcriptional rate and mRNA stability changes in pituitary tumor cells. J. Mol. Biol. 181: 41-62.

Eisenberg, S. 1984. High density lipoprotein metabolism. I. Lipid Res. 25: 1017-1058.

Friedman, J.M., E.Y. Chung, and J.E. Damell, Jr. 1984. Gene expression during liver regeneration. J. Mol. Biol. 179:3 7- 53.

Fulton, R. and G.D. Birnie. 1985. Post-transcriptional regulation of rat liver gene expression by glucocorticoids. Nucleic Acids Res. 13: 6467-6482.

Giachielli, C. and J. Omiecinski. 1986. Regulation of cytochrome p450b and p450e mRNA expression in the developing rat. Hybridization to synthetic oligodeoxyribonucleo- tide probes. J. Biol. Chem. 261: 1359-1363.

Gillespie, D. and S. Spiegelman. 1965. A quantitative assay for DNA-RNA hybrids with DNA immobilized on a membrane. J. Mol. Biol. 12: 829-842.

Greengard, O. 1977. Enzymic differentiation of human liver: comparison with the rat model. Pediatr. Res. 11: 669-676.

Groudine, M., M. Peretz, and H. Weintraub. 1981. Transcrip- tional regulation of hemoglobin switching in chicken em- bryos. Mol. Cell. Biol. 1: 281- 288.

Groudine, M. and C. Casimir. 1984. Post-transcriptional regulation of the chicken thymidine kinase gene. Nucleic Acids Res. 12: 1427-1446.

Guyette, W.A., R.J. Matusik, and J.M. Rosen. 1979. Prolactin- mediated transcriptional and post-transcriptional control of casein gene expression. Cell 17: 1013-1023.

Idzerda, R.J., H. Huebers, C.A. Finch, and G.S. McKnight. 1986. Rat transferrin gene expression: Tissue-specific regulation by iron deficiency. Proc. Natl. Acad. Sci. 83: 3723-3727.

Jefferson, L.S., W.S.L. Liao, D.E. Peavy, T.B. Miller, M.C. Appel, and J.M. Taylor. 1983. Diabetes-induced alterations in liver protein synthesis: Changes in the relative abundance of mRNAs for albumin and other plasma proteins. 1. Biol. Chem. 258: 1369-1375.

Jefferson, D.M., D.F. Clayton, J.E. Damell, Jr., and L.M. Reid. 1984. Post-transcriptional modulation of gene expression in cultured rat hepatocytes. Mol. Cell. Biol. 4:1929-1934.

Jefferson, D.M., L.M. Reid, M. Giambrone, D.A. Shafritz, and M.A. Zern. 1985. Effects of dexamethasone on albumin and collagen gene expression in primary cultures of adult rat hepatocytes. Hepatology 5: 14-20.

Kakita, K., S.J. Giddings, P.J. Rotwein, and M.A. Permutt. 1983. Insulin gene expression in the developing rat pancreas. Dia- betes 32:691-696.

Kelsey, G.D., S. Povey, A.E. Bygrave, and R.H. Lovell-Badge. 1987. Species- and tissue-specific expression of human ~ - antitrypsin in transgenic mice. Genes Dev. 1:161-171.

Koga, K., D.W. O'Keefe, T. Iio, and T. Tamaoki. 1974. Tran- scriptional control of ~-fetoprotein synthesis in developing mouse liver. Nature 252: 495-497.

Kurtz, D.T., A.E. Sippel, R. Ansab-Yiadon, and P. Feigelson. 1976. Effect of sex hormones on the level of messenger RNA for the rat hepatic protein a2u globulin. J. Biol. Chem. 251: 3594-3598.

Lamers, W.H., R.W. Hanson, and H.M. Meisner. 1982. cAMP stimulates transcription of the gene for cytosolic phos- phoenol pyruvate carboxykinase in rat liver nuclei. Proc. Natl. Acad. Sci. 79: 5137-5141.

LeBouton, A.V. 1974. Growth, mitosis, and morphogenesis of the simple liver acinus in neonatal rats. Dev. Biol. 41: 22- 30.

Leys, J.E., G.F. Crouse, and R.E. Kellens. 1984. Dihydrofolate reductase gene expression in cultured mouse cells is regulated by transcript stabilization in the nucleus. J. Cell. Biol. 99: 180-187.

Liao, W.S.L., A.R. Conn, and J.M. Taylor. 1980. Changes in rat a-fetoprotein and albumin mRNA levels during fetal and neonatal development. I. Biol. Chem. 255: 10036-10039.

Lou, C.-C., W.H. Li, M.N. Moore, and L. Chan. 1986. Structure and evaluation of the apolipoprotein multigene family. J. Mol. Biol. 187: 325-340.

Mannervik, B. 1985. The isozymes of gluthionone transferase. Avd. Enzmol. Relat. Areas Mol. Biol. 57: 347-417.

Marcu, K.B. 1987. Regulation of expression of the c-myc proto- oncogene. Bioessays 6:28-32.

Meehan, R.R., D.P. Barlow, R.E. Hil, B.L.M. Hogan, and N.D. Hastie. 1984. Pattem of serum protein gene expression in mouse visceral yolk sac and foetal liver. EMBO J. 3: 1881- 1885.

Omiecinski, C.J . 1986. Tissue-specific expression of rat mRNAs homologous to cytochrome P-450b and P-450e. Nu- cleic Acids Res. 14: 1525-1539.

Panduro, A., F. Shalaby, F.R. Weiner, L. Biempica, M.A. Zem, and D.A. Shafritz. 1986. Transcriptional switch from albumin to ot-fetoprotein and changes in transcription of other genes during carbon tetrachloride induced liver regeneration. Biochemistry 25:1414-1420.

Panduro, A., F. Shalaby, and D.A. Shafritz. 1987. Liver-specific gene expression in various pathophysiologic states. Hepa- tology 7:10S- 18S.

Paul, J., D. Conkie, and R.I. Freshney. 1969. Erythropoietic cell population changes during the hepatic phase of erythropoi- esis in the foetal mouse. Cell Tissue Kinet. 2: 283-294.

Peterson, J.A. 1976. Clonal variation in albumin messenger RNA activity in hepatoma cells. Proc. Natl. Acad. Sci. 73: 2056-2060.

Peterson, J.A. and M.C. Weiss. 1972. Expression of differentiated functions in hepatoma cell hybrids: Induction of mouse albumin production in rat hepatoma-mouse hepatoma fibroblast hybrids. Proc. Natl. Acad. Sci. 69: 571-575.

Pollard, A.M., D. Bemuau, I. Toumier, L.G. Lelgres, D. Schoe- vaert, G. Feldmann, and J.M. Sala-Trepat. 1986. Cellular analysis by in situ hybridization and immunoperoxidase of alpha-fetoprotein and albumin gene expression in rat liver during the perinatal period. J. Cell. Biol. 103: 777-786.

Powell, D.J., J.M. Friedman, A.J. Oulette, K.S. Krauter, and J.E. Damell, Jr. 1984. Transcriptional and post-transcriptional





Panduro et al.

control of specific messenger RNAs in adult and embryonic liver. ]. Mol. Biol. 179: 21-35.

Reid, L.M., M. Narita, M. Fujita, Z. Murray, C. Liverpool, and L.C. Rosenberg. 1985. Matrix and hormonal regulation of differentiation in liver cultures. In Isolated and cultured hepatocytes. {ed. A. Guillouzo and C. Guillouzo), pp. 225-258. Inserm, Paris.

Ricca, G.A., R.W. Hamilton, I.W. McLean, A. Conn, J.E. Ka- linyak, and J.M. Taylor. 1981. Rat al-acid glycoprotein mRNA. Cloning of double-stranded cDNA and kinetics of induction of mRNA levels following acute inflammation. 1. Biol. Chem. 256: 10362-10368.

Sala-Trepat, J.M.J., T.D. Dever, T.K. Sargent, S. Sell, and J. Bonner. 1979. Changes in expression of albumin and a-fetoprotein genes during rat liver development and neoplasia. Biochemistry 1 8 : 2 1 6 7 - 2 1 7 8 .

Scherer, G., W. Schmid, C.M. Strange, W. Rowekamp, and G. Schutz. 1982. Isolation of cDNA clones coding for rat tyrosine aminotransferase. Proc. Natl. Acad. Sci. 79: 7205- 7208.

Schumperli, D. 1986. Cell-cycle regulation of histone gene expression. Cell 45" 471-472.

Sellem, C.H., M. Frain, T.A. Erdos, and J.M. Sala-Trepat. 1984. Differential expression of albumin and fetoprotein genes in fetal tissues of mouse and rat. Dev. Biol. 102: 51-60.

Sul, H.S., L.S. Wise, M.L. Brown, and C.S. Rubin. 1983. Cloning of cDNA sequences for murine malic enzyme and the iden- tification of aberrantly large malic enzyme mRNA in MOD-1 null mice. J. Biol. Chem. 259: 555-559.

1984. Cloning of cDNA sequences for murine ATP-citrate lyase. J. Biol. Chem. 259: 1201-1205.

Summers, J. 1975. Physical map of polyoma viral DNA fragments produced by cleavage with a restriction enzyme from Haemophilus aegyptius endonuclease R HaeIII. I. Virol. 15: 946-953.

Tamaoki, T. and N. Fausto. 1984. Expression of the c~-fetoprotein gene during development, regeneration, and carcino- genesis. In Recombinant DNA and cell proliferation (ed. G.S. Stein and J.L. Stein), pp. 145-168. Academic Press, New York.

Thieler, K. 1972. The house mouse. Springer-Verlag, Berlin. Thomas, P.S. 1980. Hybridization of denatured RNA and small

DNA fragments transferred to nitrocellulose. Proc. Natl. Acad. Sci. 77" 5201-5205.

Tilghman, S.M. and A. Belayew. 1982. Transcriptional control of the murine albumin c~-fetoprotein locus during development. Proc. Natl. Acad. Sci. 79" 5254-5257.

Vannice, J.L., J.M. Taylor, and G. Ringold. 1984. Glucocorti- coid-mediated induction of ~l-acid glycoprotein: Evidence for hormone-regulated RNA processing. Proc. Natl. Acad. Sci. 81" 4241-4245.

Yap, S.H., R. Strair, and D.A. Shafritz. 1978. Effect of a short term fast on the distribution of cytoplasmic albumin messenger ribonucleic acid in rat liver. J. Biol. Chem. 253: 4944-4950.





10.1101/gad.1.10.1172Access the most recent version at doi: 1:1987, Genes Dev.

A Panduro, F Shalaby and D A Shafritz of liver-specific gene expression during rat development.Changing patterns of transcriptional and post-transcriptional control

References

http://genesdev.cshlp.org/content/1/10/1172.full.html#ref-list-1

This article cites 61 articles, 31 of which can be accessed free at:

License

ServiceEmail Alerting

click here.right corner of the article or

Receive free email alerts when new articles cite this article - sign up in the box at the top

Copyright © Cold Spring Harbor Laboratory Press


http://genesdev.cshlp.org/lookup/doi/10.1101/gad.1.10.1172

http://genesdev.cshlp.org/content/1/10/1172.full.html#ref-list-1

http://genesdev.cshlp.org/cgi/alerts/ctalert?alertType=citedby&addAlert=cited_by&saveAlert=no&cited_by_criteria_resid=protocols;10.1101/gad.1.10.1172&return_type=article&return_url=http://genesdev.cshlp.org/content/10.1101/gad.1.10.1172.full.pdf



changing patterns of transcript!onal and post ...genesdev.cshlp.org/content/1/10/1172.full.pdf ·...

Documents