Developmental and Tissue-specific Expression of al-Microglobulin mRNA in the Rat*

A rat liver cDNA library was constructed in the hgtll expression vector. Three clones expressing al- microglobulin, an immunosuppressive plasma protein, were detected by screening with rabbit antiserum against rat or,-microglobulin. The a,-microglobulin ac- tivity from one of the clones, 6b, was confirmed with monoclonal antibodies in a solid phase radioimmunoassay. The nucleotide sequence of the fragment (165 base pairs) was determined, and the translated amino acid sequence (55 amino acids) showed a 75% homology to human al-microglobulin (position 122-1 76). Southern blots of restriction endonuclease-digested rat DNA in- dicated two distinct genes with a,-microglobulin homology when probed with radioactive cDNA fragment from clone 6b. Northern blots showed the presence of a single mRNA species in rat liver, and the level was low in 1-month-old animals, increased to reach a max- imum during adulthood (3 months), and decreased with aging (12 months). The a,-microglobulin concentration in rat serum showed the same age dependence between 1 and 12 months, with the highest values at 3 months. Embryonic development (8.5-day to 17.5-day) was studied using total fetal RNA, and expression of al- microglobulin mRNA was detected in low amounts only at day 15.5. al-Microglobulin mRNA levels, studied by an RNA dot blot assay, were high in liver and kidney, low in brain

A rat liver cDNA library was constructed in the hgtll expression vector. Three clones expressing almicroglobulin, an immunosuppressive plasma protein, were detected by screening with rabbit antiserum against rat or,-microglobulin. The a,-microglobulin activity from one of the clones, 6b, was confirmed with monoclonal antibodies in a solid phase radioimmunoassay. The nucleotide sequence of the fragment (165 base pairs) was determined, and the translated amino acid sequence (55 amino acids) showed a 75% homology to human al-microglobulin (position 122-1 76). Southern blots of restriction endonuclease-digested rat DNA indicated two distinct genes with a,-microglobulin homology when probed with radioactive cDNA fragment from clone 6b. Northern blots showed the presence of a single mRNA species in rat liver, and the level was low in 1-month-old animals, increased to reach a maximum during adulthood (3 months), and decreased with aging (12 months). The a,-microglobulin concentration in rat serum showed the same age dependence between 1 and 12 months, with the highest values at 3 months. Embryonic development (8.5-day to 17.5-day) was studied using total fetal RNA, and expression of almicroglobulin mRNA was detected in low amounts only at day 15.5. al-Microglobulin mRNA levels, studied by an RNA dot blot assay, were high in liver and kidney, low in brain and testis, and none were found in hypothalamus and spleen cells. al-Microglobulin (al-ml) is a low molecular weight immunosuppressive plasma protein (1-3) that has been shown to impede the antigen stimulation of lymphocytes and the migration of leukocytes. aI-m is a glycoprotein (4), and its concanavalin A-reactive glycopeptides have been shown to carry some of the immunomodulatory activity of the protein (5). Immunoglobulin A binds covalently to part of the plasma cy1-m (6). Immunochemically identical homologues have been purified from guinea pig (7) and rat (8, 9) urine, and the *This work was supported in part by grants from the Swedish Medical Research Council, King Gustav V's 80-year foundation, and the Medical Faculty, University of Lund. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. protein was reported to be synthesized by human (10) and guinea pig liver (11) and rat hepatocytes (9). Contradictory results concerning the synthesis of a,-m by blood cells have been published , and this issue remains unresolved.

The nucleotide sequence(s) reported in thispaper has
However, the results so far have been obtained using immunochemical techniques, the sensitivity of which is not sufficient to detect low levels of expression, or rapidly secreted proteins. Also, the possibility of immunochemical cross-reaction poses the question of whether the detected proteins are really al-m or rather members of a family of proteins all sharing homology with al-m, and thus yielding complex results when quantitated. The size of primate al-m is larger than in non-primates (16), and, while the amino acid sequence of human al-m is known (17), nothing has yet been reported of the primary structure of the animal homologues. Thus, the differences in molecular weight remains unexplained, as does the possible existence of other ai-m-like proteins.
We have chosen to address both of these problems by isolating and sequencing a cDNA clone that encodes a portion of rat oLl-m. The radiolabeled cDNA was subsequently used for investigations into the genomic organization of el-m and possible homologues, and into the expression of cyl-m mRNA in rat liver and other tissues.

RESULTS
Isolation and Characterization of a]-m cDNA-The rat liver cDNA library was constructed in the Xgtll expression vector and had a complexity of 1.5 x IO6 plaques. Approximately 50,000 plaques were plated and screened with antibody directed against rat al-m. Three positive plaques were detected, and these remained positive throughout the rescreening process.
To ensure that the antibody binding was to al-m and not a contaminating antigen, one of the al-m positive clones, Xgtll 6b, was suspended in LB-medium and lysed. The lysate was coated onto a microtiter plate and incubated with anti-al-m antibody BN11.6 or, as a control, anti-rat K chain antibody MaR. BN11.6, but not MaR, was bound to the Xgtll6b lysate ( Fig. 1) probed with rabbit antisera to rat al-m and P-galactosidase.
The result is shown in Fig. 2. The control lysate contained Pgalactosidase with an apparent molecular weight of 114,000 ( Fig. 2 A , lane 3), while Xgtll6b also expressed a larger variant of this protein-M, = -121,000 (A, lane 2). Blotting with anti-al-m (B) showed that this protein was only present in Xgtll6b (lane 2), and that the al-m in this clone resided in a protein with a molecular weight of 121,000. Human al-m was added to the molecular weight standards (lane l ) , which were also incubated with both antisera. As expected, human al-m reacted only with anti-al-m (B, lane I), and had an apparent molecular weight of 31,000. The size of the inserts in each of the three clones was approximately 150 bp as seen by agarose gel electrophoresis after digestion with EcoRI. The insert of Xgtll 6b was subcloned into M13mpl1, and the DNA sequence was determined. There was a single open reading frame coding for 55 amino acids (Fig. 3A). A comparison of the amino acid sequence coded for by this fragment with the sequence of human al-m (17) demonstrated that there was 75% homology in the region compared (Fig. 3B). Based on this high homology, as well as the strong and competitive interaction with monoclonal and polyclonal antibodies against al-m, we thus concluded that the cDNA fragment coded for a portion of rat aI-m.
Southern Blotting-The number of genomic regions coding for al-m was determined by hybridizing the radioactively labeled al-m cDNA fragment to Southern blots of BamHI-, PstI-, HindIII-, or EcoRI-digested rat, mouse, and human DNA (Fig. 4). The results showed two regions in the rat genome with homology to al-m, one of them hybridizing consistently weaker than the other. We were unable to detect other fragments even after washing the filters under conditions of low stringency (0.2 X SSC at 42 "C, data not shown).
Only a single al-m-homologous fragment was detected in both the human and mouse genomes when DNAs from these species were probed in a similar manner.
Expression of al-m mRNA in Rats-To characterize the mRNA coding for al-m, total rat liver RNA (75 pg) was separated on agarose gels after glyoxylation and blotted to nitrocellulose. Hybridization of radioactive al-m cDNA to these Northern blots indicated a single mRNA species with a size of 1250 bp (Fig. 5). diluted 250 times with PBS + 0.05% Tween 20, and finally incubated for 1 h with 50 pl of lZ5I-protein G (1 X 10' cpm/ml). After washing and drying, the wells were counted in a y counter. Background binding of the antibodies (coating with PBS) was subtracted. The mean and standard deviation of triplicates are given. The tissue distribution of mRNA coding for al-m was examined using an RNA dot blot assay. Several dilutions of total RNA from brain, hypothalamus, kidney, liver, spleen, and testis were bound to nitrocellulose and hybridized with radioactively labeled cDNA Xgtll6b. Strongest hybridization Southern blots of genomic DNA digested with various restriction enzymes were hybridized with 32P-labeled al-m cDNA fragment. Lanes 1-4 were rat liver DNA digested with EcoRI, BamHI, PstI, and HindIII, respectively. Lane 5 was mouse liver DNA digested with PstI, and lune 6 was human peripheral lymphocyte DNA cut with BamHI. The film was overexposed to detect weakly hybridizing bands. Markers are in kilobases. Total rat liver RNA (75 pg) was denatured with glyoxal and subjected to electrophoresis on 1.5% agarose gels, blotted to nitrocellulose, and hybridized with 32P-labeled a1-m anti-sense strand DNA. The liver RNA was obtained from individuals at 2,3,4,6, or 12 months of age (lanes 1-5, respectively). Molecular weight markers were a Hind111 digest of Xgtll DNA. was seen in the liver, followed by the kidney, weak hybridization was detected in the brain and testis, and no or very little hybridization was observed in the spleen and hypothalamus (Fig. 6).
The presence of al-m mRNA primarily in the liver prompted the question of whether there were any differences in expression of al-m through the development of the animal. Total RNA was prepared from rats at various developmental stages from precisely timed early embryos (8.5-day) through late embryogenesis (17.5-day) and from the livers at selected times of postnatal and adult development. The steady state levels of al-m mRNA were tested by hybridization to Northern blots. We could not detect any al-m expression during rat embryogenesis with the exception of a small, but reproducible hybridization at day 15.5 (data not shown). During postnatal development (Fig. 5), the level of al-m mRNA in the rat liver at 2 months was increased at 3 months and then decreased gradually with aging (4,6, and 12 months). The level observed at .I month (not shown) was lower than that observed at 2  kidney (3), liver (4), spleen (5), and testis (6) was denatured with formaldehyde and applied in serial dilutions to nitrocellulose paper, and was hybridized to 32P-labeled a*-m cDNA specific for the antisense strand. Each dot contained a total amount of 20 pg of RNA composed of rat tissue RNA, at the amount given in the figure, plus yeast tRNA. , * -. months, and was close to the limits of detection. Throughout development, the size of the mRNA remained constant, and only one species of RNA was observed.
Finally, the serum concentrations of the protein al-m were measured by radioimmunoassay in rats between 1 and 12 months old. Two rats of each age, using the strain Wistar Furth (also used for mRNA analysis), yielded a mean of 8.8 (1 month), 11.8 (2 months), 15.0 (3 months), 14.9 (4 months), 12.0 (6 months), and 9.1 mg/l (12 months) al-microglobulin in serum.

DISCUSSION
Clones from the rat liver cDNA library constructed in Xgtll were screened for the expression of a protein which could bind to rabbit anti-rat al-m. Three such clones were found, and one of them, Xgtll 6b, reacted more strongly than the other two with the antiserum, as judged by the intensity of color in the horseradish peroxidase screening assay. A small possibility that the polyclonal antiserum recognized a minor impurity in the original preparation of rat al-m (9) could be excluded by showing that the hybrid protein produced in Escherichia coli also bound to the monoclonal antibody BN11.6, which we have shown to be monospecifically directed against human, guinea pig, rat, and rabbit The size of the positive cDNA clones were estimated to be about 150 bp, and the size of the encoded al-m portion of the P-galactosidase hybrid protein was determined by SDS-PAGE and Western blotting to be approximately 7 kDa. The sequence of the cDNA clone revealed an insert of 165 bp or 55 amino acids. The derived amino acid sequence of the cDNA insert showed 75% homology to human al-m in position 122-176 (17). We thus concluded that the cDNA insert in X g t l l clone 6b coded for part of rat al-m.
We have shown that primate al-m has a larger size than non-primate al-m (16). Thus, human al-m has a molecular weight of 31,000 (by SDS-PAGE) or 26,000 (by gel chromatography) (2), and rat al-m correspondingly 24,000 or 22,000 (9).* The peptide coded for by the rat al-m cDNA corresponded to about one-third of human al-m, which has a total length of 181 amino acids. Since no start or stop codon was included in the cDNA, the fragment contained neither the amino terminus nor the carboxyl terminus of native rat al-m. It is clear, however, from the sequence comparison that the difference in molecular weight between the two homologues cannot be accounted for by a deletion in the carboxyl terminus of rat al-m, since this could be maximally six amino acids. Also, by determination of the amino-terminal amino acid sequence of rat al-m, we have been able to show that no peptide was missing in the amino terminus compared to the human homologue? Hence, the size difference between the two homologues is accounted for either by (a) large gap(s) internally along the peptide or, interestingly and more likely, a difference in carbohydrate substitution.
The Southern blots suggested the presence of two DNA regions in the rat with strong homology to our al-m probe. It is very likely that these represent two different genes. If the two hybridizing bands represent two different exons of one gene: the a]-m gene, they would have to be separated by introns containing the specific nucleotide sequences for restriction enzymes EcoRI, BamHI, PstI, and HindIII. One of the bands showed a consistently weaker signal than the other when probed with al-m cDNA, suggesting that it had a lower homology to al-m. Computer searches, made at this laboratory, among proteins with known amino acid or nucleotide sequences, did not reveal any strong homologies to human a]m or the translated rat cDNA, suggesting that al-m is a unique protein which is not a member of a "protein family." One report has shown a weak similarity (18%) of human aIm to serum retinol-binding protein and the milk protein @lactoglobulin (35). This, however, is beyond the limits of detection by nucleic acid hybridization. Thus, the second DNA region detected here, assuming it is a separate gene, codes to our knowledge for an uncharacterized protein. This second gene was not observed, however, in either mouse or human DNA where only one band was detected. We have not determined whether this gene was actually missing from the genomes of these two species or whether, due to species divergence, it could no longer hybridize to our probe.
The messenger RNA coding for rat al-m was observed to be approximately 1250 nucleotides long. Since the human a]m peptide i-built of 181 amino acids, which would be coded for by 543 nucleotides, greater than half of the al-m mRNA codes for leader peptides or is nontranslated.
As expected, aI-m mRNA was expressed in the liver of rats. This confirmed earlier studies of production of rat cyl-m by hepatocytes (9). Intermediate amounts of a]-m mRNA were found in kidneys and very small amounts in brain and testes. To our knowledge, there have been no other reports of the synthesis of aI-m in either kidney, brain, or testis.
No al-m mRNA was found in spleens or hypothalami of the rats. The absence of al-m in spleen supported our earlier findings that no cyl-m could be demonstrated in association with human lymphocytes or lymphoid cell lines, using radioimmunoassay (15). However, other groups have indicated the synthesis of cyl-m by human and rat lymphocytes (12)(13)(14), using detection with antibodies. Since antibody-antigen interactions allow a higher degree of cross-reactivity than oligonucleotide hybridization under the given conditions, perhaps these antibodies, even monoclonal, bound to cross-reactive determinants, for example cell-surface carbohydrate or even the product of another gene such as the second al-mlike gene observed in the Southern blot analysis. Alternatively, the antibodies detected exogenous al-m from insufficient washings, or bound to receptors for al-m, which is known to interact with the white blood cells (3).
The expression of a]-m in embryonic rat tissue was only detected in 15.5-day-old fetuses. No al-m mRNA was found in younger or older fetuses. This agreed with the finding of Tejler (36) that the blood of human fetuses contained maximal levels of the protein at 15-17 weeks, and then rapidly decreased until birth. The postnatal expression of rat liver alm mRNA showed a surprising peak at the age of 3 months. The study was made on two separate sets of individual rats, and the results were identical. The serum concentrations of al-m, determined immunochemically, also showed this age dependence: high at 3 months and low at 1 month and 12 months. The only previous report concerning the age dependence of al-m concentrations, by Takagi et al. (37), demonstrated the opposite finding: high plasma concentration of human al-m in infancy and old age, and lower concentrations in normal adults, thus revealing a difference between these two species. Then perhaps further studies will show that the expression and plasma concentration of al-m, an immunosuppressive protein, can be correlated with the activity of the immune system and its fluctuations with age.