Biomedical subjects

E Kosik

Publications and source records attributed to E Kosik.

3 recordsLinked to original sources

PubMed1986

Design of retroviral vectors for the insertion of foreign deoxyribonucleic acid sequences into the avian germ line.

Because the available avian leukosis viral (ALV) vectors are moderately oncogenic in vivo, they are not suitable for insertion into the germ line. A significant reduction in the oncogenicity of the ALV vectors can be achieved by substituting the noncoding long terminal repeats (LTR) regions of the ALV virus with the LTR of the nononcogenic endogenous RAV-O virus. There is good evidence that the resulting RAV-O LTR vectors can be inserted into the germ line of domestic chickens and have the potential for inserting cloned sequences that can be used for poultry improvement.

Animals↗

PubMed1984

Mutagenesis of the region between env and src of the SR-A strain of Rous sarcoma virus for the purpose of constructing helper-independent vectors.

The major goal of these experiments is to derive stable, helper independent, retroviral vectors using the SR-A strain of Rous sarcoma virus. Because src is flanked by direct repeats of 110 bases, both src, and sequences that replace src in vector constructions, are lost at high frequency. We have sought to eliminate this homology in order to stabilize the vectors. One copy of the direct repeat must be retained for the virus to replicate properly. Because the downstream direct repeat is linked to the polypurine tract the entire downstream direct repeat cannot easily be eliminated. We therefore sought to eliminate the upstream direct repeat. Using linkers a series of defined deletions and duplications has been created within the region between env and src. The region is relatively large, 379 bases, and has a complex history (it is derived from three different nucleic acid segments each with a distinct and separate origin). We show here that this region provides no functions essential for growth and, for src expression, provides only a functional splice acceptor. We were able to successfully replace the splice acceptor found in the wild type virus with an unrelated splice acceptor partially derived from a synthetic DNA segment. The final product is a replication competent virus that expresses src, and that lacks the entire upstream repeat. Since src is flanked by ClaI sites in these constructions, src can easily be replaced by other genes. Substituting the Tn5 neo gene for src in this construction yields a virus that expresses the neo gene nonselectively.

Animals↗

PubMed1984

Mutation of a termination codon affects src initiation.

The four Rous sarcoma virus messages gag, gag-pol, env, and src all derive from a full-length RNA precursor. All four messages contain the same 5' leader segment. Three of the messages, gag, gag-pol, and env, use an AUG present in this leader to initiate translation. The src AUG initiation codon lies 3' of the leader segment, 90 bases downstream of the gag initiation codon in the spliced src message. However, in the spliced src message a UGA termination codon lies between the gag AUG and the src AUG. All three codons are in the same reading frame. By using oligonucleotide-directed mutagenesis, the UGA termination codon has been converted to CGA. Cells infected with the mutant (called 1057 CGA) were spindle shaped, distinct from the rounded shape of cells infected with the parental Rous sarcoma virus. The mutant virus initiates src translation at the gag AUG, producing a 63,000-dalton src protein. We suggest that the wild-type src message produces two polypeptides, a very small (nine-amino acid) peptide that is initiated at the gag AUG and the 60,000-dalton src protein that is initiated at the src AUG.

Amino Acid Sequence↗