An anthraquinone-linked duplex DNA oligomer containing 60 base pairs was synthesized by PCR. The strand complementary to the quinone-containing strand has four isolated GG steps, which serve as traps for a migrating radical cation. Irradiation of the quinone leads to electron transfer from the DNA to the quinone forming the anthraquinone radical anion and a base radical cation. The radical cation migrates through the DNA, causing reaction at GG steps revealed as strand breaks. The efficiency of strand cleavage falls off exponentially with distance from the quinone (slope ‫؍‬ ؊0.02 Å ؊1 ). This finding necessitates reinterpretation of mechanisms proposed for radical cation migration in DNA. We propose that radical cations form self-trapped polarons that migrate by thermally activated hopping.

In mammals, testis determination is under the control of the testis-determining factor borne by the Y chromosome. SRY, a gene cloned from the sex-determining region of the human Y chromosome, has been equated with the testis-determining factor in man and mouse. We have used a human SRY probe to identify and clone related genes from the Y chromosome of two marsupial species. Comparisons of eutherian and metatherian Y-located SRY sequences suggest rapid evolution of these genes, especially outside the region encoding the DNA-binding HMG box. The SRY homologues, together with the mouse Ube1y homologues, are the first genes to be identified on the marsupial Y chromosome.

We describe a new publicly available algorithm for identifying absent sequences, and demonstrate its use by listing the smallest oligomers not found in the human genome (human "nullomers"), and those not found in any reported genome or GenBank sequence ("primes"). These absent sequences define the maximum set of potentially lethal oligomers. They also provide a rational basis for choosing artificial DNA sequences for molecular barcodes, show promise for species identification and environmental characterization based on absence, and identify potential targets for therapeutic intervention and suicide markers.

ABSTRACTcDNA clones coding for chicken cartilage link protein were isolated and sequenced. The DNA sequence for the entire core polypeptide of the mature link protein and the predicted signal peptide consists of 1065 nucleotides. The deduced primary translation product (355 amino acids) has a molecular mass of 40.7 kDa; the calculated molecular mass of the mature link protein core polypeptide (340 amino acids) is 39.06 kDa. The DNA sequence contains two tandemly arranged repeat sequences that may code for repeated functional domains of link protein involved in binding to hyaluronic acid. The mRNAs for chicken link protein are 6.0, 5.8, and 3.0 kilobase pairs, and the difference between the sizes of the RNA species lies in the 3' untranslated region.Proteoglycan monomers of bovine (1-3) and chicken (4) hyaline cartilage and the Swarm rat chondrosarcoma (5) can interact with hyaluronic acid to form macromolecular aggregates. This interaction is stabilized by one or more link proteins (6)(7)(8)(9)(10). Isolated link protein affects proteoglycan aggregate structure (11) and it can bind to either hyaluronic acid (12) or proteoglycan monomer (13).Link proteins vary in size (14-18), in part as a result of differences in glycosylation (14,18). Partial amino acid sequences from Swarm rat chondrosarcoma link protein are homologous to those of bovine link protein (19,20). Whereas no amino acid sequence has been published for chicken cartilage link protein, it has been reported to be closely related to bovine link protein (21). Here we provide the complete amino acid sequence of chicken link protein deduced from cDNA sequences. MATERIALS AND METHODSIsolation of cDNA Clones. The isolation of poly(A)+ RNA from 14-day-old chicken embryo sternal cartilage, synthesis, and cloning of cDNA has been described (22). Two cDNA libraries constructed in either pUC8 or pUC9 vectors (at the Sal I-EcoRI sites) were screened with a synthetic oligonucleotide probe (GARGCNGARCARGCNAARGT) that was deduced from the amino acid sequence of link protein identical in bovine cartilage (19) and rat chondrosarcoma (20) link proteins (Glu-Ala-Glu-Gln-Ala-Lys-Val). The oligonucleotide was synthesized by the phosphoramidite method using a Microsyn (Systec, Minneapolis, MN) DNA synthesizer and was end-labeled by using [y-32P]ATP and T4 polynucleotide kinase. Filters were prehybridized at 37°C in 0.9 M NaCl/90 mM sodium citrate, pH 7/0.1% NaDod-S04/0.02% Ficoll/0.02% polyvinylpyrrolidone/0.02% bovine serum albumin/0.05% sodium pyrophosphate/10% dextran sulfate. After 6 hr, 2 x 106 cpm (20 ng) of end-labeled probe was added per 2 ml of prehybridization mixture per filter. After overnight hybridization the filters were washed four times (20 min per wash) at 370C and twice (30 min per wash) at 450C in 0.9 M NaCl/90 mM sodium citrate, pH 7/0.05% sodium pyrophosphate.DNA Sequencing. The nucleotide sequence was determined by using the method of Sanger et al. (23). Inserts of cDNA clones were subcloned in M13 phage vectors (24). The cDNA clones were linearized at...

Phosphorothioate-modified oligonucleotides were injected into pregnant female mice to assess the effect on developing embryos. Injections were carried out during two different time periods, one when embryos were in preimplantation stages of development (about 3.5 days of development) and the other after implantation, when both a fetus and placenta are present (from days 9.5 to 11.5 of development). Three different phosphorothioate-modified oligonucleotides were injected. One, which had a sequence not present in the mouse genome, was used to ask whether nonspecific toxic or teratogenic effects on embryos result from treatment of the mother. A second was complementary to the mRNA of the testis-determining factor gene Sry and was used to ask whether a specific developmental pathway (i.e., sex determination) could be disrupted in embryos in vivo. The third was the complement of the anti-Sry sequence. None of these oligonucleotides reduced the frequency of successful pregnancy after mating or the average litter size from that observed in controls animals. Furthermore, examination of 291 pups or fetuses from all oligonucleotide-injected pregnant females revealed no developmental defects regardless of which sequence was used. It is concluded that injection of phosphorothioate-modified oligonucleotides into pregnant females according to the protocols described here is not toxic or teratogenic to embryos in a nonspecific way. Also, anti-Sry oligonucleotides did not influence sex determination in embryos, although there are several possible explanations for this.