Regulation of Synthesis of Amyloid A-Related Protein

REGULATION OF SYNTHESIS OF AMYLOID A-RELATED PROTEIN*

Robert S. Stearman. Clifford A. Lowell, William R. Pearson, and John F. Morrow

Department of Molecular Biology and Genetics School of Medicine

The Johns Hopkins University Baltimore, Maryland 21205

INTRODUCTION

The concentrations of several serum proteins increase during infection, inflammation, or malignant neoplasia. These acute phase proteins include C-reactive protein, fibrinogen, haptoglobin, serum amyloid A, a,-acid glycoprotein, and a,-acute phase glycoprotein, among C-reactive protein is useful as an indicator of inflammation and may play a role in host defense. Serum amyloid A is of interest in pathology because of its antigenic and structural relationship to the major protein component of amyloid fibrils found in reactive systemic amyloid~sis.~ This is the most frequent form of amyloidosis and is a serious complication of diseases involving chronic inflammation, one of which is rheumatoid arthritis. Serum amyloid A is a possible precursor to amyloid A fibril protein. The individual polypeptide of serum amyloid A which is homologous to amyloid A is called SAAL. It is obtained by denaturation of serum amyloid A, which is a lipoprotein containing apolipoprotein A-I also.6 SAAL can increase 1000-fold or more in 20 hr after administration of bacterial endotoxin to mice, reaching 1 mg/ml.'.8

The basis for the rapid rise in concentration of acute phase serum proteins is of interest from the point of view of molecular genetics. Research on the synthesis of SAAL is facilitated by its low molecular weight, amino acid sequence informa- tion.',' and the fact that its messenger RNA (mRNA) is readily detected by in vitro translation of murine acute phase liver RNA." However, the amino acid sequence of SAAL shows microheterogeneity, indicating that SAAL is actually a family of closely related but not identical polypeptides, encoded by nonallelic genes. In the C57BL/6 inbred mouse strain there is evidence for at least four SAAL sequences, and in BALB/c and AKR, at least two.'

We have found that SAAL mRNA increases at least 500-fold in murine liver in the course of the acute phase response to lipopolysaccharide (endotoxin). Indeed, the amount of SAAL mRNA in normal liver is so low that it has not yet been possible to detect it by the methods employed. SAAL biosynthesis increases to 2.5% of total hepatic protein synthesis during the acute phase response. Cytoplas- mic actin synthesis also increases, while synthesis of serum albumin decreases to one-third of its normal rate. SAAL mRNA is tissue-specific because it was not detected in the kidney, an important site of amyloid A fibril accumulation. SAAL mRNA must be much less abundant there than in the liver, if any of this mRNA is found in the kidney at all." In this study, we have explored the genetic basis for

*This work was supported hy Grants CA16519 and GM07309 from the National Institutes of Health.

106 0077-8923/82/0389-0106 $01 75/0 ' 1982. NYAS

Stearman et a].: Amyloid A-Related Protein 107

the induction of SAAL mRNA and protein synthesis in the acute phase response.

MATERIALS AND METHODS

A recombinant phage Charon 4A "library" containing a partial EcoRI digest of murine DNA from BALB/c/3T3 cultured cells was generously provided by K. Peden, P. Mounts, D. Nathans and T.J. Kelly, Jr. Dog pancreas microsomes were the generous gift of H. Lodish. The recombinant plasmid pRS48" was labeled with 32P by nick translation and used to identify recombinant phage containing homologous DNA." Filter transfer hybridization of r2P] DNA with restriction endonuclease digests of DNA was as des~ribed.'~. '~ Minor modifications of the methods of McKnight et al.14 were used for preparation of nuclei, synthesis and purification of [3zP] RNA, and hybridization of it with recombinant plasmid DNA. Nucleotide sequence determination was according to Maxam and Gilbert." pRS48 DNA was end-labelled at one of the following sites: either the Hpa I1 endonuclease site encoding amino acids 44 & 45 (TABLE I), by polynucleotide kinase with [y3'P] ATP or Micrococcus Juteus DNA polymerase with [CU-~~P] dCTP; or at the Pst I site joining the cDNA to pBR322 (preceding codon 8) by terminal deoxynucleotidyl transferase with [c~-~*P] cordycepin 5'-triphosphate. Lipopolysaccharide was obtained from Difco Laboratories (Escherichia coli Olll:B4, Boivin method].

RESULTS

The recombinant plasmid pRS48 was identified by its ability to base-pair with SAAL mRNA." To describe it briefly, the plasmid was made by reverse transcription of murine hepatic polyadenylylated RNA purified 24 hr after lipopolysaccharide (LPS) administration. (We found that the SAAL mRNA concentration is maximal from 9 to 24 hr after intraperitoneal LPS administration.] The second DNA strand was synthesized, homopolymer dC tails were added, and the DNA was cloned at the Pst I restriction endonuclease site of plasmid pBR322 by standard methods. The recombinant plasmids which base- paired more efficiently with 32P- labeled complementary DNA (cDNA] to LPS- induced liver RNA than with DNA complementary to normal liver RNA were examined further. The plasmid DNAs were denatured, bound to nitrocellulose, and incubated with RNA from liver after LPS administration. When the RNA hybridized to plasmid pRS48 was eluted and translated in a wheat germ-derived protein synthesizing system, a 14,000 molecular weight polypeptide was produced which immunoprecipitated specifically with anti-amyloid A immunoglobulin G.

SAAL has a molecular weight of about 12,000.8.9 We have electrophoresed SAAL and the 14,000 dalton in vitro product in the same dodecyl-sulfate- polyacrylamide gel. The results indicate that SAAL is indeed about 2,000 daltons smaller. When a secreted polypeptide is synthesized in vitro in the absence of microsomes, a larger precursor with additional amino acid residues at its N-terminus (a signal peptide] is produced in almost all cases examined. We suggested that the 14,000 dalton polypeptide is such a precursor to SAAL, since CNBr cleavage (at methionine fesidues] yielded a 9400 dalton peptide from it and from SAAL." This is known to be the C-terminal peptide of SAAL,'.' indicating that the 14,000 dalton polypeptide has additional residues at its N-terminus. In

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Stearman et al.: Amyloid A-Related Protein 109

confirmation of this, we find that translation of post-LPS liver RNA in the presence of dog pancreas microsomes yields almost as much 12,000 dalton polypeptide as 14,000 dalton. Both immunoprecipitate specifically with anti- amyloid A IgG. Translation with microsomes is a general method for cleavage of signal peptides from secreted proteins.”

We have characterized plasmid pRS48 further by nucleotide sequence analy- sis.” First a number of restriction endonuclease cleavage sites within its cDNA portion were mapped. The 5’40-3‘ orientation of the mRNA sequence in the plasmid map was established by hybridization of a 5’-end-labelled DNA restriction fragment, central to the map, with LPS-induced polyadenylylated RNA. This fragment primer was then extended by reverse transcription” and the product was tested for its homology with the neighboring restriction fragments.12 The orientation found is confirmed by the amino acids encoded by the cloned cDNA (below).

Reverse transcript clones made by these methods always lack the sequences found at the 5’ ends of the mRNAs, missing at least 15 nucleotides but sometimes much more. We determined how much 5‘ mRNA sequence was missing from pRS48. A 51-nucleotide A h I restriction fragment of pRS48, starting five bases within the 5’ end of its coding sequence, was used to prime reverse transcription of post-LPS liver RNA.” The products were electrophoresed in a polyacrylamide gel containing 7M urea and appropriate DNA molecular weight standards.” The most abundant product had a length of about 190 nucleotides including primer. This indicates that pRS48 lacks about 135 nucleotides of the 5‘-terminal sequence of SAAL mRNA. pRS48 contains about 420 base pairs derived from cDNA. Consequently it may contain all of the 3’ end sequence of SAAL mRNA, since the mRNA’s length is 650 nucleotides, including a poly A terminus which may be 100 nucleotides.”

The N-terminal sequences of SAAL and amyloid A (AA) have been determined for several mouse ~ t r a i n s . * ~ ~ ~ ~ ~ For comparison, we determined the nucleotide sequence of the corresponding region of pRS48. An unambiguous sequence was obtained by use of the method of Maxam and Gilbert on each of the DNA strands.“ The sequence at the extreme 5‘ end of the coding strand is shown in the top line of TABLE 1, and the amino acids it encodes are shown in the second line. Termination codons are present in both of the other reading frames. The sequence aligns well with SAAL and AA polypeptide sequences, starting at residue 8 of murine SAAL.’.’ Remarkable is a stretch of 13 amino acids with perfect homology to all known AA sequences (residues 32-44), even including duck and mink AA (TABLE 1). Twenty-one amino acids encoded can be compared to murine SAAL and AA sequences, because only the N-terminal 28 residues have been determined for these polypeptides. Fifteen of 21 (71%) of the amino acids match. The homology of these amino acids encoded by pRS48 is actually better with monkey AA (16 of 21, 76%). The entire sequence shown is 84% and 82% homologous with the corresponding 38 amino acids of monkey and mink AA, respectively (TABLE 11.

The differences between the residues encoded by pRS48 and known AA sequences are not randomly distributed. They occur more frequently at positions which differ among AA polypeptides of different species of animals, i.e., at sites less conserved during evolution. A notable example is position 27, where the pRS48 sequence translation differs from the murine SAAL and AA residues but encodes the asparaginyl residue found in all other known SAAL or AA species (TABLE 11. Similar findings occur at positions 14 and 21, where pRS48’s translation differs from murine AA but agrees with the monkey and human AA residues land

110 Annals New York Academy of Sciences

mink as well, at residue 21). The positions at which various species' AA polypeptides differ most are 10 and 14, and here the pRS48 translation differs from murine SAAL. However, it agrees with duck AA and with human and monkey AA at these positions, respectively. The serine encoded at position 13 and lysine at 25 have not been reported in SAAL or AA. However, the DNA encoding residues 13 and 14 is an Xba I restriction endonuclease site found in the corresponding mouse gene (see below] and in pRS48. There is evidence that murine SAAL comprises a family of related but distinct polypeptide sequences; we suggest that pRS48 encodes one of these.

pRS48 was synthesized using hepatic mRNA of a Swiss mouse as template." We have isolated the BALB/c genome segment homologous to pRS48. Recombi- nant phage, containing partially digested EcoRI endonuclease fragments of BALB/c/3T3 DNA, were screened for those homologous to pRS48 by plaque transfer hybridization." One of 660.000 plaques screened bound ["PI pRS48 DNA efficiently. The phage is called XSAA1. It contains a nucleotide sequence identical to that shown in TABLE 1. This suggests that genetic differences among mice are not the cause of the differences between the protein sequence encoded by pRS48 and the murine SAAL sequence.

To determine whether XSAAl's murine DNA fragments are faithful copies of the mouse genome sequences homologous to pRS48, we determined the sizes of restriction fragments of both which hybridize with pRS48, using filter transfer hybridization." The BALB/c mouse liver DNA restriction fragments which hybridize most efficiently with ["PI pRS48 are: after EcoRI digestion, 7.0 kb (thousands of base pairs]; Bgl 11, 4.6 kb; Xba I, 1.1 kb; and Pst I, 3.8 kb. The fragments of XSAAl which hybridize correspond to the respective fragments above after EcoRI, BgJ I1 or Xba I digestion. The Pst I fragment of XSAAl which hybridizes with pRS48 is not 3.8 kb, but mapping experiments show that it contains phage vector DNA. The pRS48 sequence is near the right end of the murine DNA in XSAAl. Only one of the Pst I sites bounding it in the mouse genome is in this clone. The sites for Eco RI, Bgl I1 and Xba I, yielding the above fragments, have been mapped within the cloned mouse DNA. The data indicate that XSAAl contains a faithful copy of the mouse DNA most homologous to pRS48.

An interesting finding in these experiments is that pRS48 also hybridizes to other mouse DNA fragments, though less efficiently. For example, after Eco RI digestion, fragments of 9.0, 6.0 and 3.1 kb bind pRS48 DNA, in addition to the major band at 7.0 kb. Additional minor bands of mouse DNA are also found with homology to pRS48 after Bgl 11, Pst I, and Xba I digestion. The vector plasmid pBR322 does not hybridize significantly with mouse DNA. It is not possible to interpret the number of homologous mouse genes from the number of homologous restriction fragments because of the prevalence of noncoding intervening sequences within eukaryotic genes. The results are nevertheless suggestive of two or more homologous genes, complementary to pRS48, in the haploid genome of the inbred BALB/c strain.

The DNA sequence shown in TABLE 1 is present in the corresponding region of XSAA1, as determined by chemical sequence methods." Significant is the Xba I site encoding residues 13 and 14, found in pRS48, in XSAA1, and in the mouse gene. This indicates that codons 13 and 14 (see above) are not artifacts introduced by cloning methods. XSAAl also contains DNA encoding the 3' end of the mRNA corresponding to pRS48, separated by an intron of about 250 base pairs from the exon containing Table 1's sequence.


The quantities of hepatic mRNAs encoding SAAL and q-acid glycoprotein increase greatly in the acute phase resp~nse.~. '~ The resulting increase in synthesis of these proteins can account for their elevated serum concentrations. To understand the basis of the acute phase response, we have carried out a series of experiments exploring the mechanism of the increase in SAAL mRNA.

Isolated nuclei incubated with ribonucleoside triphosphates synthesize RNA species in quantities generally representative of those made in the tissue and physiological state from which they were obtained.14 We have used this method to test whether the transcription of genes homologous to pRS48 increases in the acute phase response. At various times after LPS administration, murine liver nuclei were isolated and incubated 45 min. at 26" with [CX-~~P] UTP. The amount of labeled RNA complementary to pRS48 was measured by hybridization with an excess of denatured plasmid DNA (TABLE 2). Filters with an equal amount of the bacterial plasmid vector pBR322 served as controls in the same incubation.

TABLE 2 SYNTHESIS OF SAAL RNA BY ISOLATED NUCLEI A ~ E R ENDOTOXIN ADMINISTRATION*

Radioactivity Incorporated UTP Endotoxin (pmol/mg. Input Probe Bound (cpm'l Transcription

Administration DNA) (cpm. x 10'1 pBR322 pRS48 Rate I x 10'1

None 19 1.3 225 & 65 296 k 110 50 k 90 3 hr 17 2.7 122 * 14 911 r 10 290 f- 30 6.5 hr 16 1.0 102 f 2 648 2 41 550 f 64 9 hr 14 0.9 98 t 6 480 * 52 420 k 90 12 hr 13 3.4 134 f 17 355 25 60 f 14

*Mice received 50 pg. lipopolysaccharide intraperitoneally. At the indicated times, liver nuclei were isolated and incubated with [alpha-3zP] UTP. 770 Curies/mmol." RNA was purified as described but without acid precipitation. It ranged in size from 100 to 800 nucleotides pedominantly. with a mean of about 200 nucleotides. The [32P]-RNA from nuclei containing about 150 pg. of DNA was incubated with filters containing 2 pg. denatured, linear plasmid DNA in 50% formamide. 0.6M NACI. O.12M Tris-HC1 pH 7.0.8 mM EDTA, 20 pg/ml each poly (rA) and poIy [rC), 20 pg/mI yeast tRNA, and 0.2% SDS. Hybridizations were carried out for 32 hr. at 45°C. Filters were washed and incubated with RNase A as described. The results shown in counts/min. [cpm) are the mean standard deviation for 5 filters (normal liver nuclear RNA) or 2 filters (RNA from liver nuclei after endotoxin administration]. Transcription rate is the radioactivity bound to each pRS48 filter minus that bound to pBR322, divided by the total RNA input radioactivity per filter (shown in the third column).

Slightly more RNA bound to pBR322 filters than to those with no DNA [not shown).

The pRS48 filters did not bind significantly more RNA from normal liver nuclei than did the control filters (TABLE 2). Consequently, the data do not show any transcription of genes homologous to pRS48 in normal liver nuclei.

However, more ["PI RNA was bound by the pRS48 filters than by the bacterial vector filters at all times from 3 to 12 hr after LPS administration. The transcription of these genes is highest 6.5 to 9 hr. after LPS administration (TABLE 2). It is lower at 12 hr. [and also at 24 hr.; data not shown). This transcription reaches a peak as early as SAAL mRNA does, or earlier. SAAL mRNA concentration shows


a high plateau from 9 to 24 hr. and declines to 25% of the maximum level by 43 hr." The transcription of RNA homologous to pRS48 begins to decline much earlier.

We have examined the methylation of cytosine residues in genes homologous to pRS48 to determine whether it changes as the transcription does. Within a number of genes, methylation is correlated with absence of transcription, when various tissues of vertebrates are compared. Methylation appears to cause inactivity of some integrated retrovirus genesz4 and metallothionein genesz5 We have used the technique which has been most widely applied to detect methylation, the digestion of DNA by either Hpa I1 or Msp I restriction endonuclease. Both cleave at CCGG sequences in DNA. Hpa I1 cannot cleave if the second C is methylated, while Msp I cleaves CCGG sites regardless of the state of methylation of the second C residue. The vast majority of methylation in vertebrate DNA is on C residues 5' to a G. A defect of the method is that many CG sequences do not occur in Hpa I1 sites. However, in the cases examined, the differences in methylation have occurred at several Hpa I1 sites in both introns and exons of genes and in the neighboring DNA as well.

DNA from various BALB/c murine tissues was digested with an excess of either Msp I or Hpa I1 endonuclease, electrophoresed in a 1% agarose gel to separate fragments according to molecular weight, and transferred to a nitrocellulose filter.''.'3 The fragments homologous in sequence to recombinant plasmid pRS48 were then located by annealing with ["PI pRS48 DNA. As expected, the Msp I fragments detected are similar in DNA from all tissues used: normal liver, kidney, brain and spleen, and liver 6 hr and 24 hr after LPS administration. Fragments of 7, 2.1. 1.9, 1.5, 0.44 and 0.38 kb are detected by ["PI pRS48 binding, and a 1.7 kb fragment in some DNA preparations. The 1.5 and 0.44 kb fragments correspond in size to Hpa I1 fragments of XSAAl which anneal with pRS48. They presumably result from digestion of the mouse gene cloned in XSAAl. The other fragments detected may result from digestion of other genes homologous to pRS48 in the BALB/c mouse genome (see above). The fragments listed are reproducibly found in comparable intensities in all Msp I digests and do not appear to reflect partial digestion.

The DNA fragments binding pRS48 after Hpa I1 digestion are also similar among the tissues listed above, but differ from the M s p I results in a way that indicates extensive methylation of these DNA sequences. Most of the ["PI pRS48 bound to Hpa I1 fragments is at 4.0 kb and in a continuous distribution of fragments larger than 7 kb. The amount of ["P] DNA found at the positions of the Msp I fragments listed above is 3070 or less after Hpa I1 digestion, compared to either the Msp I digests of the same DNA preparations or to the larger Hpa I1 fragments detected. The only exception is spleen DNA, where about half of the ["PI DNA bound to 2.1, 1.9, 1.5, 0.44 and 0.38 kb fragments after Hpa I1 digestion and about half bound to larger. methylated DNA fragments. After LPS administration there is little change in methylation of liver DNA homologous to pRS48 (tested by Hpa I1 digestion 6 hr. or 24 hr. after LPS).

Methylation of DNA near the 5'-end coding region may be more significant for control of transcription. To investigate this, a ["PI cDNA copy of the 5' end of mRNA homologous to pRS48 was prepared. The 51-nucleotide A h I fragment of pRS48, used above, was annealed with post-LPS liver polyadenylylated RNA and used to prime reverse transcription." The products contain up to 135 nucleotides of ["PI cDNA specific for the 5'-end sequence. These products were annealed with transfer filters similar to those tested with pRS48. The unusual result is that very little difference exists between the Msp I fragments and the Hpo 11


fragments detected by homology to this 5' end [$'PI cDNA. The major fragments detected are 7,2.5,2.2,1.9 and 1.7 kb after M s p I or Hpa I1 cleavage of DNA from the tissues used. LPS administration did not alter the results from liver DNA.

DISCUSSION

We have described a recombinant plasmid, pRS48, which is able to base-pair with SAAL mRNA.'' A partial nucleotide sequence of pRS48 is reported here. It encodes a polypeptide with strong homology (71%) to the N-terminal region of murine SAAL and AA.8.9.20 Beyond residue 28, where the published murine polypeptide sequence ends, pRS48 encodes residues strongly homologous to human, monkey, mink and duck amyloid The homology is perfect with residues 32 through 44 of all these AA proteins. This region must be crucial for an important function of SAAL in vertebrate physiology, since its sequence has been conserved since the divergence of the ancestors of mammals and birds, about 270 million years ago. In this time, 8% to 27% of the amino acid residues of insulin and globins have been changed by mutation, and 49% to 62% of the residues of proinsulin C peptides have changed.26

Heterogeneity of SAAL or AA amino acid sequence has been reported in inbred mice, in duck and in man.8-18,19 pRS48 may encode a form of SAAL whose sequence has not been reported for one of the following reasons. This form may have a blocked N-terminus, as reported for about half of the termini of murine AA." Alternatively, it may occur at a low concentration because it is synthesized at a low rate or is cleared rapidly from plasma. It is also possible that this form may not be efficiently purified by current methods. The gene corresponding to pRS48 is transcribed, since the plasmid was synthesized by reverse transcription of mRNA. More research is needed to choose among these alternatives.

Mammalian cells are able to regulate mRNA synthesis at steps other than transcription. Changes in RNA termination and processing alter the ratios of adenovirus-2 mRNAsZ7 However, most of the control of cellular gene expression appears to occur at the level of RNA synthesis. This is what the data in this report indicate for genes homologous to pRS48, encoding amyloid A-related protein, during the acute phase response. The synthesis of RNA homologous to pRS48 is very rapid 6.5 hr after LPS administration. It is three times higher as a proportion of RNA produced than the highest value found among 16 liver RNA sequences in a recent study." Of course the RNA hybridizing with pRS48 may be transcribed from more than one homologous gene. The transcription of this RNA increases quickly, reaching half-maximal rate only 3 hr after LPS administration (TABLE 2). This transcription reaches its peak earlier and declines earlier than the total amount of SAAL mRNA, which has a broad plateau from 9 to 24 hr and declines then." The transcription time course measured using pRS48 is essentially that expected for synthesis of SAAL mRNA precursors. No increase was observed in total RNA synthesis by isolated nuclei after LPS administration.

The data do not demonstrate any transcription of SAAL genes in normal liver cell nuclei. Future studies may permit a lower limit of detection and a more precise estimation of the magnitude of the increase in SAAL RNA synthesis in the acute phase response.

The data indicate little or no methylation of Hpa I1 endonuclease sites near the 5'-ends of mouse genes homologous to pRS48. Also, little change is found in methylation near the interior and 3' ends of these genes during a time when their transcription increases greatly. In contrast, three sites in or near the ovalbumin


gene are predominantly methylated in tissues other than oviduct and predominantly unmodified in mature oviduct tubular gland cells.29 The acute phase transcription rate of genes homologous to pRS48 is comparable to the rate of the fully induced ovalhumin gene. Clearly the correlation of methylation with transcription is much less for SAAL genes than for ovalbumin, indeed there may be no such correlation for SAAL. In a cultured fibroblast cell line, methyl groups modifying DNA cytosine residues are stable, not actively removed.30 Hepatocytes and other cells may also be incapable of removing DNA methyl groups in a short time, e.g., the 3 hr in which the transcription measured here reaches a high rate. A limitation of these studies is that methylcytosine residues outside CCGG sequences cannot be detected by Hpa I1 and Msp I cleavage. Hence these enzymes only examine a subset of all the cytosine residues within a gene. Subject to this reservation, the results suggest that phenomena other than DNA methylation control SAAL gene transcription in the acute phase response.

SUMMARY

The concentration of serum amyloid A polypeptide (SAAL) increases greatly during the acute phase response to infection or inflammation. We find that SAAL synthesis comprises 2.5Yr of murine hepatic protein synthesis after lipopolysaccharide (LPS] administration, but much less in normal liver. SAAL messenger RNA (mRNA) in liver increases at least 500-fold above the normal level. A recombinant plasmid homologous to SAAL mRNA has been isolated, as has most of the mouse genome DNA encoding the plasmid’s nucleotide sequence. This gene is transcribed into RNA much more frequently after LPS administration than it is in normal liver. In a number of other mammalian genes, cytosine methylation is inversely related to the rate of transcription. Methylation of CCGG sequences in hepatic DNA homologous to the recombinant plasmid has been examined. Little or no change is found after LPS administration. This suggests that other factors are responsible for the increase in SAAL mRNA in the acute phase response.

ACKNOWLEDGMENTS

We thank M. A. Kahler and C. G. Peltzman for assistance.

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Regulation of Synthesis of Amyloid A-Related Protein