Tissue specific expression of mouse transferrin during development and aging

Mechanisms of Ageing and Development, 56 (1990) 187--197 Elsevier Scientific Publishers Ireland Ltd.

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TISSUE SPECIFIC EXPRESSION OF MOUSE TRANSFERRIN DURING DEVELOPMENT AND AGING

FUNMEI YANG a'*, WILLIAM E. FRIEDRICHS a, JAMES M. BUCHANAN a, DAMON C. HERBERT a, FRANK J. WEAKER~, JEREMY H. BROCKb and BARBARA H. BOWMAN ~ "Department of Cellular and Structural Biology, The University of Texas Health Science Center, San Antonio, TX 78284 (U.S.A.) and bUniversity Department of Bacteriology and Immunology, Western Infirmary, Glasgow G I I 6NT (U.K.J (Received July 2nd, 1990)

SUMMARY

Transferrin (TF) is a major plasma protein that binds ferric iron and transports it to all target tissues of the body. This study is the first step to identify the tissue specific expression of the transferrin gene in mice during development, into maturity and throughout the aging process. The transferrin gene expresses mainly in mouse liver, the cerebral hemispheres and cerebellum. In mouse, transferrin is expressed in peritoneal macrophages and in mouse macrophage cell line MO59. At 19 days of gestation, transferrin mRNA is detected in the fetal lung, heart, stomach and kidney. TF mRNA levels increase in liver throughout gestation with maximum expression occurring at 19 days. Transferrin mRNA was detected in placentas of pregnant mice, with levels progressively increasing throughout the term of pregnancy. The levels of liver TF mRNA in mouse vary in a cyclic manner during the development increasing with the aging processes. Because of the dynamic nature of tissue requirements for transferrin during homeostasis the TF gene serves as a promising system for analyzing tissue-specific regulation in vivo during development and aging. Results from this study designate periods in the life-span of the mouse where regulatory mechanisms interacting with the TF gene appear to dynamically alter its expression.

Key words: Transferrin; Gene expression; Mouse; Development; Aging; mRNA

*To whom all correspondence should be addressed. Abbreviations: ALB, albumin; TF, transferrin; kb, kilobases; PBS, phosphate buffered saline. 0047-6374/90/$03.50 Printed and Published in Ireland

© 1990 Elsevier Scientific Publishers Ireland Ltd.

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INTRODUCTION

Transferrin (TF) is a major glycoprotein of vertebrate plasma. Its role is to carry ferric iron from the intestine, reticuloendothelial system and liver parenchymal cells to erythroid precursors and proliferating cells in the body. Transferrin is the product of an ancient intragenic duplication that led to homologous carboxyl and amino domains, each of which binds one ion of ferric iron. Transferrin is a plasma protein of biological interest, not only because of its evolutionary history, but also because of its role as a growth factor required for the proliferation of normal and malignant cells [1,2]. The evolution and genetic regulation of transferrin expression have recently been reviewed [3]. Transferrin is required for differentiation of mouse fetal kidney [4], muscle [5] and lymphocytes [6,7]. Provision of transferrin is also critical for proper development of teeth [8]. Like most plasma proteins, the TF gene is expressed mainly in the liver; however important extra-hepatic sites of transferrin expression also have been discovered in the vertebrate central nervous system [9--11], testis sertoli cells [12], mammary gland [13], lymphocytes [7] and the thymus gland [14]. Lower levels of transferrin have been found to be synthesized in rat heart, spleen, kidney and muscle [9]; these levels seem to vary during different stages of development. Little data are available regarding the extra-hepatic expression of mouse transferrin during development, in maturity and throughout the aging process. The aim of this study is to analyze expression of the TF gene in liver and in extra-hepatic tissues during the life-span of the mouse in order to detect alterations of gene expression accompanying development and aging in vivo. The cellular signals responsible for the regulation of transferrin expression are not understood but can be predicted to involve metal, hormonal, autocrine and mitotic factors that act in trans upon regions of DNA of the TF gene to control gene expression positively and negatively. The results of this study have provided new information about TF expression in mammals (1) specifying extra-hepatic sites of TF mRNA transcripts in thymus, epididymis, adrenal glands, uterus and ovary; (2) comparing TF mRNA levels in tissues derived from fetal, young, middle aged and aged mice; (3) describing the progressively increasing levels of TF mRNA in the placenta during pregnancy and (4) demonstrating the presence of TF mRNA in mouse macrophages. MATERIALS AND METHODS

Isolation o f R N A s

Organs and tissues of C57BL/6J male mice were studied. Tissues were stored in liquid nitrogen until RNA isolation was carried out. Peritoneal macrophages were isolated as described previously [15]; mice were injected intraperitoneally with 2 ml of Brewer thioglycollate medium (Difco Cat. No. 0236-01-5; prepared according to the instructions on the bottle) 4 days before use. The animals were sacrificed with CO 2 and a volume of 2.5 ml of cold sterile PBS was injected into the peritoneal cav-

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ity of the mice to gently rinse out the cells. The fluid was then withdrawn and centrifuged. The cell pellet was resuspended in RPMI 1640 (Sigma R5382) plus 1007ofetal calf serum and was placed in a tissue culture petri dish and incubated for 2 h. Adherent cells were collected and RNA was isolated. The human promonocytic cell line U937 was obtained from the Beatson Institute for Cancer Research, Glasgow and MO59, a murine hybridoma made by fusing mouse spleen cells with the murine macrophage-like cell line P388DI [16] was provided by Dr. Michael Fischbach, UTHSC-SA. Cells were routinely cultured in RPMI 1640/10070 fetal calf serum. Logarithmic phase cells were collected by centrifugation and used for RNA extraction. In cases where only small amounts of tissues or cells could be obtained from a mouse, samples of tissues were collected and pooled from several age-matched animals. Total cellular RNA was extracted by lysing the ceils or homogenizing the tissues in guanidinium thiocyanate followed by phenol-chloroform extraction [17]. Poly(A÷) RNA was obtained by fractionating total RNA using oligo(dT) cellulose chromatography [18]. The C57BL mice were obtained from the National Institute of Aging (provided by Charles River Laboratory, contractor). The animals were maintained by the contractor in a pathogen-free environment. The survival rate of C57BL mice under these conditions was reported by the contractor to be 5007o survival to 28 months of age, 25°70 survival to 32 months of age and 10°70 survival to 33 months of age. Northern blot analysis Northern blot analysis was performed as described by Fourney et al. [19], with a modified procedure for formaldehyde agarose gel electrophoretic separation of RNA [20,21]. Preparations of RNA loaded on 1°70 agarose gels were electrophoresed at 4 volts/cm gel length for 6 h and transferred to Nitroplus 2000 filters (Micron Separation Inc.) in 10 x SSC at room temperature. The filter was baked at 80°C under vacuum and prehybridized in 50°/o formamide, 5 x SSPE, 5 × Denhardt and 250/ag/ml denatured E. coli DNA at 37 °C for 3--5 h. The filter was then hybridized at 37°C overnight in the same solution plus 10070dextran sulfate and the 32p-labeled probe. Human TF cDNA insert [22] was labeled with 32p as described by Feinberg and Vogelstein [23]. After hybridization, filters were washed and autoradiographed [19]. The RNA blots were analyzed by densitometric quantitation (Quick Scan R & D, Helena Laboratories). Relative intensities of the hybridization signals were calculated with the aid of a GS 370 program (Hoefer Scientific Instruments). Filters were stripped in 50~70 formamide, 10 mM Tris (pH 7.5), 1 mM ethylene diamine tetra-acetic acid (EDTA), 0.1 °7o sodium dodecyl sulfate (SDS) for 2--4 h at 65 °C before hybridization with a second probe. RESULTS AND DISCUSSION

In vertebrates the liver synthesizes and secretes transferrin into the circulation, where it. contributes to the homeostasis of the organism by binding ferric iron and

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transporting it to target tissues throughout the body. After entering the cell by receptor mediated endocytosis transferrin delivers iron to a non-lysosomal, acidic compartment within the cell and returns to extracellular regions to carry out its function of iron delivery. Synthesis o f transferrin is regulated by cytokines, hormones, iron stores and inflammation. An extra-hepatic supply of transferrin is often advantageous to cells in specific localities of the organism. Five non-hepatic tissues in mouse displayed high levels of expression of the TF gene. Figure 1 demonstrates by Northern analysis the mRNA levels in liver, brain, ovary, placenta and uterus. The cerebral hemispheres and cerebellum of the adult brain produced approximately 5-10070 of the TF mRNA found in liver of mice 6 months old (designated as adult liver, hereafter). The placenta after 19 days of gestation produced as much TF mRNA as found in adult liver. Expression of TF in ovary and uterus was approximately I0% that of adult liver. Low levels of mouse TF mRNA were observed in other organs examined. Figure 2 demonstrates low levels of TF mRNA in epididymis, adrenal glands (lanes 3 and

I

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M.W. Kb

lip

:e

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.,t 7.4 .,,,4.4

.42.3 1.3

Fig. 1. TF expression in m o u s e liver, brain and female reproductive organs. Poly(A ÷) R N A isolated from liver, cerebrum, cerebellum, ovary, uterus and a full term (19 day) placenta (lanes 1--6) of 2 - - 3 - m o n t h old male and female mice were analyzed by Northern hybridization using 32p-labeled h u m a n TF cDNA as a probe. The a m o u n t o f poly(A*) R N A loaded on each lane was the same (10/ag) for all the samples except ovary for which 5.8/ag was loaded. This autoradiograph represents a 24 h exposure of the film to the Northern filter.

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9 I011 1 2 1 3 1 4

M.W. Kb

~7.4 -1,4.4

Fig. 2. Northern blot analysis of TF mRNA in mouse organs. RNA preparations are derived from various organs of 2--3-month-old male and female mice. Samplesloaded on the left panel gel are 10 pg poly(A÷) RNA from seminal vesicles, muscle, epididymis, adrenal glands, submaxillaryglands (lanes 1--5) and 1 ~g poly(A*)RNA from liver (lane 6). Right panel of gel contains 10 ~g poly (A÷)RNA from thymus (lane 7), spleen, stomach, lung, heart, kidney (lanes 9--13) and 0.5 ~g poly(A÷)RNA from liver (lane 14). Lane 8 is 10 big poly(A-) RNA isolated from spleen. A lane containing liver RNA was included in each gel for the purpose of quantitative comparison. The left (lanes 1--6) and right (lanes 7--14) panels of autoradiographs werethe results of 24 and 72 h exposure, respectively.

4), thymus, spleen, lung, heart and kidney (lanes 7, 9, 11, 12 and 13). The concentrations o f m R N A s in these tissues are approximately 1°70 or less that of adult liver. Whole testes also demonstrated low levels o f TF m R N A in Northern analysis (data not shown). Trace amounts of TF m R N A were found in seminal vesicles, muscle, submaxillary gland and stomach (lanes 1,2,5 and 10). A small amount o f liver m R N A was included (1 pg in lane 6 on the left panel and 0.5 ~g in lane 14 on the right panel) for quantitative comparison. The autoradiograph on the right panel was a result of long exposure o f the Northern filter to f'dm. Minimal transcription of the TF gene may represent autocrine expression, "leaky expression" or reflect expression by cells, such as macrophages, migrating within the tissues. Macrophages collected from the peritoneal cavities o f adult mice were analyzed and found to produce TF m R N A levels corresponding to about 2 to 3°70 o f the liver levels in the same animals. Figure 3 demonstrates TF m R N A in mouse macrophages and the mouse macrophage hybridoma MO59 (lanes 2 and 4). In contrast, the human promonocytic cell line U937 did not appear to produce significant levels o f the characteristic 2.3 kb TF m R N A species; however, this line did make a low molecular weight m R N A (approximately 0.2 kb) that hybridized with human TF eDNA (Fig. 3, lane 6). The 0.2 kb fragment was repeatedly detected in different R N A preparations derived f r o m U937 cells and did not appear to result f r o m non-specific deg-

192 3

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~-

Kb

-,2.3

02

Fig. 3. Detection of TF mRNA in macrophages. Northern blot analysis was performed with poly(A*) RNA from peritoneal macrophages of 2--3-month-old mice (1.2 tag, lane 2), mouse macrophage line MO59 (10 tag, lane 4) and human histiocytic (macrophage) lymphoma line U937 (10 tag, lane 6). Lanes 1, 3 and 5 are 10 tag poly(A-) RNA from mouse macrophages, MO59 and U937 cells. The films were exposed for one week. A small molecular weight fragment (0.2 kb) detected in U937 cells hybridized to human TF cDNA.

r a d a t i o n o f R N A p r e p a r a t i o n s as e v i d e n c e d b y h y b r i d i z a t i o n results using o t h e r genes, those e n c o d i n g a l k a l i n e p h o s p h a t a s e a n d actin, as h y b r i d i z a t i o n p r o b e s . Prel i m i n a r y studies o f h u m a n p e r i t o n e a l m a c r o p h a g e s have also failed to detect synthesis o f T F (R. O r i a a n d J . H . B r o c k , u n p u b l i s h e d o b s e r v a t i o n s ) . T F m R N A c o u l d n o t be d e t e c t e d in m o u s e l y m p h o c y t e s , either with o r w i t h o u t s t i m u l a t i o n with c o n c a n a v a l i n - A , b u t was f o u n d in the m o u s e T cell line C 6 L V when e x a m i n e d b y p o l y m e r a s e c h a i n r e a c t i o n ( d a t a n o t shown). P r e v i o u s studies f r o m o u r l a b o r a t o r y h a d r e p o r t e d synthesis o f T F b y the T4 ÷ i n d u c e r subset o f h u m a n T l y m p h o c y t e s in an a u t o c r i n e p a t h w a y l i n k e d to the I L - 2 / I L - 2 r e c e p t o r a u t o c r i n e l o o p [7]. T h u s expression o f T F b y cells i n v o l v e d in the i m m u n e system m a y v a r y a c c o r d i n g to species. H a u r a n i et al. [24] have p r e v i o u s l y r e p o r t e d t h a t lysates o f m o u s e p e r i t o n e a l m a c r o p h a g e s synthesized t r a n s f e r r i n a n d p r o p o s e d t h a t i r o n released f r o m d e g r a d e d e r y t h r o c y t e s inside m a c r o p h a g e s c o u l d p r e f e r e n t i a l l y b i n d to this source o f t r a n s f e r rin a n d thus be t r a n s p o r t e d b a c k into the c i r c u l a t i o n .

193 Transcripts o f m R N A were analyzed in fetal tissue during development. TF m R N A was detected in fetal stomach, heart and lung during the 19th day o f gestation (Fig. 4, lanes 3,4 and 5). The levels found in these tissues were approximately 0.5--1070 that in adult liver. Small amounts of TF m R N A were detected in fetal kidney of the 19th day of gestation and in fetal lung of the 16th day of gestation (Fig. 4, lanes 2 and 11, respectively). Non-hepatic expression of the TF gene during the prenatal period was reported by Levin et al. [25] in rat. In muscle and other non-hepatic, non-nervous tissues of the rat, TF gene expression was reported to be maximal just before birth, then to decrease markedly during postnatal development, whereas in results reported here, non-hepatic expression in the mouse remains at approximately the same level after birth. In early postimplantation mouse development, transferrin gene expression occurs on the 7th day of gestation [26]. After 10 days of embryogenesis transferrin synthesis is detected in the visceral yolk sac where its levels increase with gestational age. Transferrin can be detected in fetal serum and in amniotic fluid as soon as these fluids accumulate [26]. During mouse gestation the TF m R N A in liver progressively increased. In the 16th day o f gestation, mouse TF m R N A levels were 20070 that of adult liver (Fig. 4, lane 12). By the 19th day of gestation the TF m R N A levels were approximately the same as in adult liver (Fig. 4, lane 7). In the placenta, synthesis of TF m R N A was followed by Northern analysis throughout pregnancy. TF m R N A in placenta at the 12th day of pregnancy was

1

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R

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11 12 13 14

Fig. 4. TF expression in mouse fetal tissues and placentas of different gestational stages. Northern blot analysis was conductedwith 5 pg total RNA from mouse fetal livers and 10/~gtotal RNA from other fetal tissues and placentas. Lanes 1--8 contain RNAs from fetal tissues after 19 days of gestation: fetal brain, kidney, stomach, heart, lung, limb buds, liver and placentas. Lanes 9--14 contain RNAs from fetuses of 16-daysgestation: fetal brain, heart, lung, liver, limb buds and maternal placentas. Lane 15 contains the 12-daygestation maternal placenta RNA. Two fragments other than the characteristic 2.3 kb TF mRNA are seen in this experiment and may result from cross-hybridization of the TF probe with ribosomal RNA.

194

about 1--2070 that o f the adult liver, and at 16 days of pregnancy was approximately 2507o the concentration of TF mRNA in adult liver. Immediately before birth (19th day of pregnancy) the TF mRNA in placenta reached almost the same level as that of adult liver. These results are shown in Fig. 4 (lanes 15, 14 and 8, respectively) and demonstrate the incremental expression of the TF gene in the placenta. Yeoh and Morgan [27] reported the synthesis of rat transferrin by combined fetal membranes at the 13th day of gestation. Aldred et al. [9] demonstrated a low level of rat TF mRNA in the placenta. The role o f transferrin could be related to the placenta's function in providing regulatory and nutritional factors, such as iron, for support of the fetus. Fetal iron is derived from maternal transferrin [28]. Iron transfer occurs from maternal transferrin binding to receptors on the maternal face of the placenta. The transferrin is then internalized and the iron released to the cell, after which the transferrin is recycled to the maternal plasma [28]. This report demonstrates an additional contribution of transferrin by the placenta that increases during pregnancy, probably as a result o f hormonal regulation. The greatest extra-hepatic contribution of rat transferrin had previously reported to be made in the brain by the oligodendrocytes [10] and the choroid plexus [9]. In developing mouse brains, TF mRNA is not detectable even at the 19th day o f gestation (Fig. 4). An increase of TF mRNA levels occurs after birth and during early development with a maximum level found between 1 and 2 months after birth, at which time the brain level is 5--10°70 that of the TF mRNA levels of adult liver (data not shown). Similar results were found in rat brains by Levin et al. [25], who reported rat transferrin mRNA to be very low before birth, then to increase gradually during the post-natal development and to reach a plateau in the adult with a concentration of 1 / 7 - - 1 / 1 0 that observed in adult liver. By 28 months, mice have advanced well into the aging process. The concentrations o f TF and ALB mRNAs in liver of mice from 1 to 28 months are shown in Fig. 5. The temporal expression of the mouse TF gene during development and aging was surprising in its cyclic nature. The highest levels of mRNA in livers of age-matched mice were found at 1 month and 28 months. The quantitation of TF mRNA in each age-matched group was obtained after scanning with the densitometer by dividing the integration value of the TF mRNA band by the average integration value obtained from TF mRNA bands in the 6-month-old mice. At 28 months the mouse TF mRNA increases 1.5-fold. A similar increase is not seen in ALB mRNA. It is interesting to compare the temporal increase of TF mRNA with other plasma proteins that have been studied during the aging process. Haptoglobin mRNA-values increase 2-fold in mice 16 months old and increased as much as 4-fold after 18 months [29]. Since in mice, transferrin and haptoglobin are both positive acute phase reactants, it is possible that the observed increases in the two mRNA levels during aging are results of age-related inflammation which was not evident pathologically. Rutherford et al. [30] demonstrated that another positive acute phase reactant, al-acid glycoprotein mRNA also increases slightly between 2 and 29 months in

195

200

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0 II

100

m

ALB TF

O > °~

m

G) rr

tip 1

4

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Age (Months)

Fig. 5. Comparison of the liver TF raRNA levels of six different age groups of male C57BL mice. Five micrograms of poly (A÷)RNA prepared from mouse livers derived from 1, 4, 6, 12, 16, and 28-month-old mice were used for Northern blot analysis. The same Northern filters were stripped and hybridized with rat albumin cDNA probe for comparison purposes. Autoradiographs with short exposures were examined to ascertain that the signals were within the linear range. The intensities of the hybridization signals were scanned with a densitometer and quantitated as described in the methods section. The mean from each group of mice (3--6 animals) was plotted against the mean from the 6-month-old mice, which was designated as 100%. Error bars represent standard error of the means. The means and error bars were computed from the data of three experiments. *P-value < 0.05 when compared to values of 6-month-old animals.

aging F i s c h e r rats. R o y a n d C h a t t e r j e e [31] r e p o r t e d changes in levels o f 17 h e p a t i c p r o t e i n s d u r i n g the aging o f rats; five p r o t e i n s increased a n d 12 d e c r e a s e d in levels as the a n i m a l s aged. S o m e o f t h e o b s e r v e d differences in gene expression o c c u r in the t r a n s c r i p t i o n step a n d a r e a t t r i b u t e d to d e c r e a s e d levels o f m R N A s [32,33]. It is n o t k n o w n at the present t i m e w h e t h e r t h e o b s e r v e d c h a n g e s in T F m R N A levels are d u e to m R N A stability o r increases in t r a n s c r i p t i o n r a t e o f the T F gene. It will be a p p l i c a b l e t o a n u n d e r s t a n d i n g o f the aging process to d e t e r m i n e the basis o f altered p l a s m a p r o t e i n gene e x p r e s s i o n d u r i n g senescence. M o u s e , b u t n o t h u m a n , t r a n s f e r r i n acts like a positive a c u t e p h a s e r e a c t a n t ; t h a t is, it increases in c o n c e n t r a t i o n a f t e r i n f l a m m a t i o n . W h e t h e r a g e - r e l a t e d changes o b s e r v e d in the T F gene expression in m o u s e s h a r e the s a m e m o l e c u l a r m e c h a n i s m s involved in t h e response to the a c u t e p h a s e r e a c t i o n is u n k n o w n . T h e i n f l a m m a t o r y a n d aging regu-

196 lations m a y be i n d e p e n d e n t in some p r o t e i n genes. Sierra et al. [34] reported a specific sequence o f D N A in o n e o f the t r a n s c r i p t i o n a l start sites o f the rat T - k i n i n o g e n gene that is specific for increased expression o f this gene d u r i n g aging but distinct f r o m a D N A sequence n e a r b y that is associated with the p r o t e i n ' s increase d u r i n g inflammation. I n s u m m a r y , results f r o m this study have p o i n t e d out periods in the life-span o f the mouse, when levels o f T F m R N A are altered, p r o b a b l y as a result of factors i n d u c e d d u r i n g d e v e l o p m e n t a n d aging a n d by v a r i a t i o n s o f h o r m o n e s a n d iron stores. F u t u r e studies are a i m e d at i d e n t i f y i n g the r e g u l a t o r y m e c h a n i s m s responsible for m o d u l a t i o n o f t r a n s f e r r i n expression d u r i n g d e v e l o p m e n t a n d aging. ACKNOWLEDGEMENTS This study was s u p p o r t e d by N I H grants A G O 6 8 7 2 a n d GM33298. We t h a n k Betty Russell for p r e p a r a t i o n o f the m a n u s c r i p t a n d graph. We t h a n k R o d Cupples for excellent technical assistance. REFERENCES

1 D. Barnes and G. Sato, Methods for growth of cultured cells in serum-free medium. AnalBiochem., 102 (1980) 255--270. 2 P.A. Seligman, Structure and function of the transferrin receptor. Prog. Hematol., 13 (1983) 131-147. 3 B.H. Bowman, F. Yang and G.S. Adrian, Transferrin: evolution and genetic regulation expression. In E.W. Casparri and J. Scandalios (eds.) Advances in Genetics, Academic Press, Orlando, 1988, pp. 1--38. 4 P. Ekblom, I. Thesleff, L. Saxen, A. Miettinen and R. Timpl, Transferrin as a fetal growth factor: acquisition of responsiveness related to embryonic induction. Proc. Natl Acad. Sci. USA, 80 (1983) 2651--2655. 5 R.L. Beach, H. Popiela and B.W. Festoff, Specificity of chicken and mammalian transferrins in myogenesis. CellDiff., 16 (1985) 93--100. 6 J.H. Brock, The effect of iron and transferrin on the response of serum-free cultures of mouse lymphocytes to concanavalin A and lipopolysaccharide. Immunology, 43 (1981) 387--392. 7 J.B. Lum, A.J. Infante, D.M. Makker, F. Yang and B.H. Bowman, Transferrin synthesis by inducer T lymphocytes. J. Clin. Invest., 77 (1986) 841--849. 8 A.M. Partanen, I. Thesleff and P. Ekblom, Transferrin is required for early tooth morphogenesis. Differentiation, 2 7 (1984) 59--66. 9 A.R. Aldred, P.W. Dickson, P.D. Marley and G. Schreiber, Distribution of transferrin synthesis in brain and other tissues in the rat. J. BioL Chem., 262 (1987) 5293--5297. 10 B. Bloch, T. Popovici, M.J. Levin, D. Tuil and A. Kahn, Transferrin gene expression visualized in oligodendrocytes of the rat brain by using in situ hybridization and immunohistochemistry. Proc. NatL Acad. Sci. USA, 82 (1985) 6706--6710. 11 I.R. Dickson and M.K Bagga, Changes with age in the non-collagenous proteins of human bone. Connect. Tissue Res., 14 (1985) 77--85. 12 M.K Skinner and M.D. Griswold, Sertoli cells synthesize and secrete transferrin-like protein. J. BioL Chem., 255 (1980) 9523--9525. 13 E.H. Morgan, Plasma protein turnover and transmission to the milk in the rat. Biochim. Biophys. Acta, 154 (1968) 478--487.

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J.B. Lum, Visualization of mRNA transcription of specific genes in human cells and tissues using in situ hybridization. BioTechniquea, 4 (1986) 32--40. J.H. Brock, I. Esparza and A.C. Logic, The nature of iron released by resident and stimulated mouse peritoneal macrophages. Biochim. Biophys. Acta, 797 (1984) 105--111. T. Uchida, S. Ju, A. Fay, Y. Liu, M.E. Doff, Functional analysis of macrophage hybridomas. 1. Production and initial characterization. J. Immunol., 134 (1985) 772--778. P. Chomczynski and N. Sacchi, Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem., 162 (1987) 156-- 159. H. Aviv and P. Leder, Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose. Proc. Natl. Acad. Sci. USA, 69(1972) 1408--1412. R.M. Fourney, J. Miyakoshi, R.S. Day, III and M.C Paterson, Northern blotting: Efficient RNA staining and transfer. Bethesda Rea. Labs. Inc. Focus, 10 (1988) 5--7. H. Lehrach, D. Diamond, J.M. Wozney and H. Boedtker, RNA molecular weight determinations by gel electrophorcsis under denaturing conditions, a critical reexamination. Biochemistry, 16 (1977) 4743--4751. D.A (joldberg, Isolation and partial characterization of the Drosophila alcohol dehydrogenase gene. Proc. Natl. Acad. Sci. USA, 77(1980) 5794--5798. F. Yang, J.B. Lum, J.R. Me(Jill, C.M. Moore, S.L Naylor, P.H. van Bragt, W.D. Baldwin and B.H. Bowman, Human transferrin: eDNA characterization and chromosomal localization. Proc. Natl. Acad. Sci. USA, 81 (1984) 2752--2756. A.P. Feinbcrg and B. Vogelstein, A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem., 132 (1983) 6--13. F.I. Haurani, A. Meyer and R. O'Brien, Production of transfcrrin by the macrophage. J. ReticuIoendothel. Soc., 14 (1973) 309--316. M.J. Levin, D. Tuil, (3. Uzan, J.C. Dreyfus and A. Kahn, Expression of the transferrin gene during development of non-hepatic tissues: high level of transferrin mRNA in fetal muscle and adult brain. Biochem. Biophys. Re8. Commun., 122 (1984)212--217. E.D. Adamson, The location and synthesis of transferrin in mouse embryos and teratocarcinoma cells. Dev. Biol., 91 (1982) 227--234. (J.C. Yeoh and E.H. Morgan, Albumin and transferrin synthesis during development in the rat. Biochem. J., 144 (1974) 215--224. H.J. McArdle and E.H. Morgan, Transferrin and iron movements in the rat conceptus during gestation. J. Reprod. Fertil., 66(1982) 529--536. B.H. Bowman, F. Yang and (J.S. Adrian, Expression of human plasma protein genes in aging transgenic mice. BioEssays, 12 (1990) 317--322. M.S. Rutherford, C.S. Baehler and A. Richardson, Genetic expression of complement factors and alpha l-acid glycoprotein by liver tissue during senescence. Mech. Ageing Dev., 35 (1986) 245--254. A.K. Roy and B. Chatterjee, Sexual dimorphism in the liver. Annu. Rev. Physiol., 45 (1983) 37-50. A.K Roy, B. Chatterjee, W.F. Demyan, B.S. Milin, N.M. Motwani, T.S. Nath and M.J. Schiop, Hormone and age-dependent regulation of alpha 2u-globulin gene expression. Recent. Prog. Horm. Res., 39 (1983) 425--461. A. Richardson, M.S. Roberts and M.S. Rutherford, Aging and gene expression. In M. Rothstein (ed.) Review o f BiologicalResearch in Aging, Vol. 2, A.R. Liss, Inc., New York, 1985, pp. 395-419. F. Sierra, (J.H. Fey and Y. Guigoz, T-kininogen gene expression is induced during aging. Mol. Cell Biol., 9 (1989) 5610--5616.