Fragments of amplified Xenopus laevis DNA, coding for 18S and 28S ribosomal RNA and generated by EcoRI restriction endonuclease, have been linked in vitro to the bacterial plasmid pSCl01; and the recombinant molecular species have been introduced into E. coli by transformation. These recombinant plasmids, containing both eukaryotic and prokaryotic DNA, replicate stably in E. coli. RNA isolated from E. coli minicells harboring the plasmids hybridizes to amplified X. laevis rDNA.Recombinant DNA molecules constructed in vitro from separate plasmids (1, 2) by the joining of DNA fragments having cohesive termini (3, 4) generated by the EcoRI restriction endonuclease (5, 6) can form biologically functional replicons when introduced into Escherichia coli by transformation (7). The E. coli tetracycline resistance plasmid, pSC101 (1, 8) (molecular weight 5.8 X 106), is useful for selection of recombinant plasmids in E. coli transformants, since insertion of a DNA segment at its single EcoRI cleavage site does not interfere with expression of its tetracycline resistance gene(s) or with the replication functions of the plasmid (1, 2).This report describes the in vitro linkage of pSC101 and eukaryotic DNA cleaved by EcoRI endonuclease, and subsequent recovery of recombinant DNA molecules from transformed E. coli in the absence of selection for genetic properties expressed by the eukaryotic DNA. The amplified rDNA (coding for 18S and 28S ribosomal RNA) of Xenopus laevis was used as a source of eukaryotic DNA, since it has been well characterized and can be isolated in quantity (9, 10). Recombinant plasmids containing both X. laevis and pSC101 DNA replicate stably in E. coli, where they are capable of synthesizing RNA complementary to X. laevi8 rDNA. MATERIALS AND METHODSDNA coding for ribosomal RNA of X. laevis, isolated by CsClgradient centrifugation, and 82P-labeled 18S and 28S X. laevis ribosomal RNA were the generous gifts of Dr. D. D.Brown. Bacterial strains and the tetracycline resistance plasmid pSC101 have been described (1, 2, 8). Covalently-closed circular plasmid DNA was isolated as described (8, 11), or Abbreviations: rRNA, ribosomal RNA; rDNA, amplified DNA containing the genes for 18S and 28S rRNA; EcoRI, the RI restriction and modification host specificity of E. coli controlled by the fi + plasmid, pHB1. (20) and E. coli ligase (the generous gift of Drs. P. Modrich and I. R. Lehman) (21) have been described. E. coli-X. laevis recombinant plasmids were constructed in vitro as follows: the reaction mixture (60 .A) contained 100 mM Tris-HCO (pH 7.5), 50 mM NaCl, 5 mM MgCl2, 1.0 jgg of pSC101 plasmid DNA, 2.5,g of X. laevis rDNA, and excess EcoRI restriction endonuclease (1 ,l, 2 units). After a 15-min incubation at 370, the reaction mixture was placed at 630 for 5 min to inactivate the EcoRI endonuclease. A 3-,ul sample was examined by electron microscopy to assess digestion. The remainder was refrigerated at 0.50 for 24 hr to allow association of the short cohesive termini; melting temperature (Tm) was ...

Major changes in the mRNA population of murine liver occur after administration of bacterial lipopolysaccharide, an agent that causes increases in the concentrations of acutephase serum proteins. The mRNA for one of these, serum amyloid A, is increased at least 500-fold compared to the normal level. It becomes one of the most abundant hepatic mRNAs, and serum amyloid A synthesis comprises about 2.5% of total hepatic protein synthesis in the acute-phase response. Its synthesis is tissue-specific in that amyloid A mRNA was not detected in the kidney, an important site of amyloid fibril accumulation. The protein synthesized in largest amount by acute-phase liver tissue in culture is cytoplasmic actin. Its relative rate ofsynthesis is increased about 5-fold compared to the normal tissue; that of serum albumin is decreased to about one-third ofits normal rate. The concentration of mRNA for serum albumin is decreased by a similar amount. Starting with induced liver RNA, we have constructed a recombinant plasmid containing most ofthe DNA sequence encoding the serum amyloid A polypeptide.

The RI restriction endonuclease of Escherichia coli converts covalently-closed circular Simian Virus 40 (SV40) DNA to unit-length linear duplex molecules. Cleavage occurs at a unique site, since denaturation and renaturation of these linear molecules yield linear but no circular molecules. The distance from the cleavage site to the SV40 DNA sequence contained in the adenovirus-SV40 hybrid, Ad2 +ND1, is 0

S, nuclease, the single-strand specific nuclease from Aspergillus oryzae can cleave both strands of circular covalently closed, superhelical simian virus 40 (SV40) DNA to generate unit length linear duplex molecules with intact single strands. But circular, covalently closed, nonsuperhelical DNA, as well as linear duplex molecules, are relatively resistant to attack by the enzyme. These findings indicate that unpaired or weakly hydrogen-bonded regions, sensitive to the single strand-specific nuclease, occur or can be induced in superhelical DNA. Nicked, circular SV40 DNA can be cleaved on the opposite strand at or near the nick to yield linear molecules. S, nuclease may be a useful reagent for cleaving DNAs at regions containing single-strand nicks. Unlike the restriction endonucleases, S, nuclease probably does not cleave SV40 DNA at a specific nucleotide sequence. Rather, the sites of cleavage occur within regions that are readily denaturable in a topologically constrained superhelical molecule. At moderate salt concentrations (75 mM) SV40 DNA is cleaved once, most often within either one of the two following regions: the segments defined as 0.15 to 0.25 and 0.45 to 0.55 SV40 fractional length, clockwise, from the EcoR, restriction endonuclease cleavage site (defined as the zero position on the SV40 DNA map). In higher salt (250 mM) cleavage occurs preferentially within the 0.45 to 0.55 segment of the map.

The simian virus 40 (SV40) DNA segment in the nondefective adenovirus 2-SV40 hybrid, Ad2+ND4, is colinear with the segment between 0.11 and 0.59 SV40 fractional length from the site at which the R, restriction endonuclease cleaves SV40 DNA. This specifies the region of the SV40 DNA molecule which induces the early SV40 antigens: U antigen, tumor specific transplantation antigen, and T antigen. A variant of Ad2+ND4, called Ad2+ND4del, was found which has a deletion of the DNA segment between 0.50 and 0.57 SV40 fractional length from the R, endonuclease cleavage point.

Restriction endonuclease EcoRI introduces double-strand staggered cleavages at unique sites in a variety of DNAs (1, 2). Cleavage at a unique position on the genomes of simian virus 40 (3, 4) and polyoma virus (5) has provided an electron microscopic basis for assessing bidirectional replication from a unique initiation site on these DNAs (6, 7).Although the chromosomal DNA of mammalian cells also replicates in a bidirectional mode (8, 9), the mtDNA purified from mouse LD cells appears to replicate unidirectionally (10, 11). There are two possible modes of unidirectional replication in which a growing fork can move either to the left or the right of a particular origin at which initiation has occurred. The mode of unidirectional replication that is operative in circular dimeric mtDNA was not defined by earlier studies (10-12) and may not be equivalent to the mode of replication in monomeric mtDNA. The replication of monomeric mtDNA has previously been assumed to proceed uni- Homoduplexes of EcoRI treated mtDNA were constructed (5, 14) and the renaturation conditions were chosen to permit reassociation of approximately 40% of the largest singlestrands in a particular sample (5). The samples were then prepared for electron microscopy as described above.

The acute phase response is characterized by changes in the serum concentrations of many proteins. A 1000-fold increase in the concentration of serum amyloid A (SAA) protein occurs within 24 hours of LPS injection in the mouse. We have isolated a cDNA clone and its corresponding genomic phage for a third, previously unreported SAA protein. The sequence of the cDNA, the gene's exons and neighboring DNA are presented along with the mapping evidence supporting the gene structure.

Contamination of platelet concentrates remains a significant clinical problem. Septic episodes may be reduced by transfusion of platelets with shorter storage intervals.