Chemical synthesis of DNA oligomers containing cytosine arabinoside.

The solid phase phospite triester synthesis of oligodeoxynucleotides containing cytosine arabinoside (araC) is described. A protected araC phosphoramadite was prepared for the introduction of araC residues at 5'termini and internucleotide positions in DNA oligomers. These oligomers were utilized to demonstrate the formation of correct 3'-5' linkages, to test for alkaline lability at the araC site, and to study the stability of duplexes containing araC-G base pairs. For the introduction of araC residues at 3' terminal positions, a protected derivative of araC was coupled to functionalized silica. This material was used to prepare a test oligomer which was characterized enzymatically.

Sequence and expression of Tangier apoA-I gene.

We have isolated and characterized the apoA-I gene from a lambda L47.1 genomic library constructed with DNA obtained from the lymphocytes of a Tangier disease patient. The DNA-derived protein sequence of Tangier apoA-I was found to be identical to normal apoA-I. Transfection of mouse C127 cells with a recombinant vector containing the Tangier apoA-I gene (pSV2-gpt apoA-I) allowed selection of stable clones resistant to aminopterin and mycophenolic acid. Analysis of these clones for apoA-I synthesis showed that the protein secreted by cells expressing the Tangier apoA-I gene was indistinguishable from the apoA-I secreted by HepG2 cells. These experiments establish that the Tangier apoA-I gene is structurally normal. It appears that the molecular basis of Tangier disease is not related to apoA-I structure or regulation of expression, but rather to other factors pertinent to apoA-I and high-density lipoprotein metabolism.

Complementary DNA derived structure of the amino-terminal domain of human apolipoprotein B and size of its messenger RNA transcript.

In this paper the sequence of a 5.2-kilobase (kb) cDNA covering the amino-terminal domain of human apolipoprotein B-100 (apoB-100) is reported. The cDNA-derived protein sequence provides the primary structure of 1748 amino acids. This segment of apoB-100 is more hydrophilic than hydrophobic and contains short stretches of predicted helical and beta structures that are interrupted by beta turns. Blotting analysis of RNA isolated from fetal human and adult monkey tissues and various human cell lines showed synthesis of apoB mRNA by liver and intestine and by cells of hepatic (HepG2) and intestinal (Caco-2) origin. The isolation and characterization of overlapping cDNA clones, which provide a nearly full-length copy of human apoB-100, are also reported. From the length of these clones the size of the cytoplasmic apoB mRNA is estimated to be 14.0 kb and codes for a protein of approximately 512,000 daltons.

Cloning of random-sequence oligodeoxynucleotides.

Methods are described for cloning random or highly degenerate nucleotide (nt) sequences. The procedures use synthetically derived mixtures of oligodeoxynucleotides (oligos) whose heterogeneous central portions are bounded at their 5' and 3' ends by sequences recognized by restriction endonucleases. Oligo collections of defined length and nt composition are synthesized by utilizing appropriate concentrations of all four nucleotide precursors during each addition step for the central region. Single-stranded oligos with appropriate 5' and 3' ends can be ligated directly, although inefficiently, into double-stranded (ds) DNA molecules with complementary 5' and 3' extensions produced by restriction endonuclease cleavage. A more general and efficient method is to convert the oligo into a ds form by incubating it with the Klenow (large) fragment of Escherichia coli DNA polymerase I. If the 3' ends are palindromic, two oligo molecules will serve as mutual primers for polymerization. The resulting products are ds molecules containing two oligo units separated by the original 3' restriction site and bounded at each end by the original 5' restriction site. After appropriate restriction endonuclease cleavage, oligo units can be cloned by standard procedures. Analysis of 26 recombinant M13 phages indicates that the nt sequences of the cloned oligos are in good accord with what was expected on a random basis.

Sequence of cDNA encoding human insulin-like growth factor I precursor.

Somatomedins (SM) or insulin-like growth factors (IGF) constitute a heterogeneous group of peptides with important growth-promoting effects in vitro as well as in vivo. Amino acid sequences have been determined for only two of them, IGF-I and IGF-II, which are highly homologous. IGF-I, which is identical with SM-C, is composed of 70 amino acid residues and IGF-II contains 73 amino acids and may be identical with SM-A. Other peptides with different charge properties but with similar SM-like or insulin-like behaviour in biological and receptor assays, have been described but have not yet been fully characterized. The liver is known to be a major site of production of these peptides, but many other tissues--especially in the fetus--may synthesize them as well. We report here the nucleotide sequence of a human liver cDNA encoding the complete amino acid sequence of IGF-I. The IGF-I coding region is flanked by sequences encoding an amino-terminal peptide of at least 25 amino acid residues and a carboxyl-terminal peptide of 35 amino acids. This provides evidence that IGF-I is synthesized as a precursor protein and that formation of IGF-I from this precursor requires proteolytic processing at both ends.

Identification and DNA sequence of a human apolipoprotein E cDNA clone.

cDNA clones encoding human apolipoprotein E were identified by screening an adult human liver cDNA library with an oligonucleotide probe. The probe was a mixture of synthetic 14-base long DNA oligomers constructed to correspond to all possible codons for apo-E amino acids 218-222. Plasmids from four of the 20 clones selected by this screening procedure were digested with PstI and all had five internal PstI sites with a total length of the cDNA insert of approximately 900 base pairs. DNA sequence analysis of one of these clones, designated pE-301, revealed that it corresponded to apo-E amino acids 81-299, and contained a standard termination codon, polyadenylation signal, and poly A tail. The DNA sequence examined included the known apo-E polymorphic sites at amino acids 112, 145, and 158, and the mutant apo-E phenotypes can all be explained on the basis of a single base substitution in the first position of each of these codons. This work supports the hypothesis that the apo-E polymorphism is due to mutations in the region of DNA coding for the apo-E structural gene.

Isolation and characterization of cDNA clones for human apolipoprotein A-I.

We have isolated cDNA clones encoding human apolipoprotein (apo) A-I. Twenty putative apo A-I cDNA clones were selected by screening 10,000 clones of an adult human liver cDNA library with an oligonucleotide probe. The probe was a mixture of synthetic 14-base-long DNA oligomers constructed to correspond to the codons for apo A-I amino acids 105-109. Four of these clones were examined further and showed 600- to 800-base-pair (bp) inserts. Preliminary restriction mapping and partial DNA sequence analysis indicated that the shorter inserts were a subset of the longer DNA inserts. DNA sequence analysis of the clone with an insert of approximately equal to 600 bp, designated pAI-113, revealed that it contained a DNA sequence corresponding to apo A-I amino acids 94-243. The DNA base sequence of this clone also contained a standard termination codon, polyadenylylation signal, and poly(A) tail. Partial DNA sequence of a second clone that contained an 800-bp insert, designated pAI-107, showed that it corresponded to apo A-I amino acids 18-243 and also included the 3' untranslated region. Isolation of these cDNA clones will facilitate molecular analyses of apolipoproteins in normal and disease states.

Chemical synthesis of two deoxyribododecanucleotides for the attachment of restriction termini to an artificial minigene.

In order to permit in vivo cloning of an artificial minigene designed to code for a modified S-peptide, the phosphodiester method for the chemical synthesis of two dodecadeoxyribonucleotides is described. Each of the latter possesses antiparallel complementarity to one of the two minigene strands and to the single-stranded EcoRI-generated end. They can thus serve as cohesive termini ("splints") for polynucleotide ligase joining.

Structure of a defective simian virus 40 genome bearing an operator from bacteriophage lambda.

We examined further the physical structure of the simian virus 40 (SV40) and bacteriophage lambda DNA sequences in an SV40-lambda hybrid that had been propagated in monkey kidney cells. The SV40 vector portion of the hybrid, which was a small fragment isolated from a reiteration mutant of SV40, contained the site for initiation of SV40 DNA replication. Electron microscope heteroduplex and restriction endonuclease analyses revealed a tandem duplication of the SV40 vector segment linked to a 2,300-base pair portion (lambda map units 71 to 76) of the lambda immunity region. The defective hybrid genome thus harbors two origins for SV40 DNA replication in addition to the leftward operator and the N gene of lambda.

Construction and propagation of a defective simian virus 40 genome bearing an operator from bacteriophage lambda.

A 2400 base pair DNA segment containing the leftward operator (OL) of phage lambda was covalently joined in vitro to a fragment of simian virus 40 (SV40) DNA harboring the SV40 replication origin. The recombinant molecule was propagated in the presence of helper wild-type SV40 DNA in monkey kidney cells and partially cloned by an infectious center procedure. After propagation in monkey cells and purification, the hybrid DNA could be distinguished from wild-type SV40 DNA by its shortened length (about 80% that of SV40), specific hybridization to denatured lambda DNA immobilized on filters, specific affinity for lambda repressor, and preservation of a large part (about 2300 base pairs) of the lambda immunity region as determined by restriction nuclease cleavage patterns and electron microscopic heteroduplex analysis. These results indicate that defective SV40 replicons can serve as vectors for propagating foreign DNA in mammalian cells.

Propagation of a segment of bacteriophage lamda-DNA in monkey cells after covalent linkage to a defective simian virus 40 genome.

A 520 base pair DNA segment was excised from the bacteriophage lamda-genome by cleavage with the bacterial restriction endonuclease, endo R. Hindll. This segment was covalently joined in vitro to an 880 base pair simian virus 40 (SV40) DNA segment which contains the initation site for SV40 DNA replication. The latter segment was derived from the genome of a defective reiteration mutant of SV40 also by endo R. Hindlll cleavage. When the recombinant molecule, together with wild-type SV40 DNA as helper, was introduced into monkey cells by DNA infection, replication of the lamda-DNA sequences was observed, and hybrid genomes were encapsidated into progeny SV40 virions. The structure of the lamda-DNA segment after serial passage in monkey cells was examined by use of restriction endonucleases and electron microscopic heteroduplex analysis.

Synthesis of deoxyguanosine polyphosphates and their interactions with the guanosine 5'-triphosphate requiring protein synthetic enzymes of Escherichia coli.

A chemical synthesis of deoxyguanosine analogs of the guanosine polyphosphates accumulated by bacteria during the stringent response is described. Both deoxyguanosine 3'-diphosphate 5'-triphosphate (d-pppGpp) and deoxyguanosine 3'-diphosphate 5'-diphosphate (d-ppGpp) were prepared, as well as the by-products deoxyguanosine 3'-monophosphate 5'-triphosphate (d-pppGp) and deoxyguanosine 3'-monophosphate 5'-diphosphate. A significant difference between d-(p)ppGpp and guanosine 3'-diphosphate 5'-tri- or 5'-diphosphate (p)ppGpp) is that the 3'-pyrophosphate moiety is alkali stable in the deoxyguanosine and alkali labile in the guanosine polyphosphates. The new GTP analogs d-pppGp and d-pppGpp were compared to GTP, dGTP, and pppGpp in their ability to support reactions catalyzed by the Escherichia coli protein synthetic enzymes initiation factor 2, elongation factor Tu, and elongation factor G (EF-G). Like pppGpp, both d-pppGp and d-pppGpp showed substantial deficiency only in reactions requiring EF-G. While d-pppGpp closely resembled pppGpp in its very low activity with EF-G, d-pppGp was somewhat more active. Nevertheless, d-pppGp was a poor substrate in EF-G-dependent translocation. Qualitatively and quantitatively its support of translocation was very similar to the reaction driven by periodate-oxidized and borohydride-reduced GTP, a derivative of GTP in which the ribose ring has been cleaved between the 2'- and 3'-hydroxyl groups.

Construction of a double-stranded deoxyribonucleotide sequence of 45 base pairs designed to code for S-peptide 2-14 of bovine ribonuclease A.

An artificial DNA duplex, each strand consisting of 45 monomers, is constructed from chemically synthesized deoxyriboöligonucleotides. The resulting bihelical polymer may code for a modified S-peptide of Ribonuclease A. This is the first synthetic duplex designed to code for a eukaryotic message.

Lambda phage DNA: joining of a chemically synthesized cohesive end.

The chemically synthesized dodecamer d(pA-G-G-T-C-G-C-C-G-C-C-C) was annealed, and was covalently joined to lambda phage DNA with bacterio-phage T(4) ligase. The 5'-end of the dodecamer was joined to a deoxyguanosine residue. Repair with DNA polymerase I established that the position of joining was the left-hand end of lambda DNA. This is the first time that a chemically synthesized oligonucleotide has been covalently joined to a naturally occurring DNA molecule.