Cysteine mutagenesis was used to test the proximity of 16 residues to protein-ligand interaction sites in maltoporin (LamB protein). LamB protein with additional cysteines was incorporated into the outer membrane of Escherichia coli except with a Ser-30----Cys substitution. Phage Lambda and starch binding was assayed before and after incubation of mutants with six thiol-specific reagents. Four categories of mutation were recognized on the basis of phenotype and modification for each of the Lambda- and starch binding sites. The thiol modification experiments helped to clarify whether the phenotype of a mutation was due to a substitution at the binding site or an indirect perturbation of the structure. This study suggests that the cysteine mutagenesis/thiol modification approach may be usefully applied to the operational mapping of surface-accessible binding sites or epitopes.

The Sloan-Kettering viruses (SKVs) are replication-defective retroviruses that transform avian cells in vitro. Each of the three SKV isolates is a mixture of viruses with genomes ranging in size from 4.1 to 8.9 kilobases (kb) with a predominant genome of 5.7 kb. Using a cDNA representing a sequence, v-ski, that is SKV specific and held in common by the multiple SKV genomes, we generated a restriction map of the 5.7-kb SKV genome and molecularly cloned a ski-containing fragment from SKV proviral DNA. Southern hybridization and sequence analysis showed that the cloned DNA fragment consisted of the 1.3-kb ski sequence embedded in the p199a9 sequence and followed by the remaining 5' half of the gag gene and small portions of both the pol and env genes. A large deletion encompassing the 3' half of gag and the 5' 80% of pol was mapped to a position about 1 kb downstream from the 3' ski-gag junction. To determine whether the cloned ski sequence had transforming activity, the ski-containing fragment and a cloned Rous-associated virus 1 (RAV-1) genome were used to construct an analog of the 5.7-kb SKV genome, RAV-SKV. Cotransfection of chicken embryo cells with RAV-SKV and RAV-1 yielded foci of transformed cells whose morphology was identical to that induced by the natural SKVs. The transformed transfected cells produced transforming virus with a 5.7-kb ski-containing genome and synthesized a gag-containing polyprotein of 110 kilodaltons (kDa). Several nonproducer clones of RAV-SKV-transformed cells were analyzed, and most were found to synthesize a 5.7-kb SKV RNA and a 110-kDa polyprotein. One clone was found to contain an 8.9-kb SKV RNA, and this clone synthesized a 125-kDa polyprotein. Since both the 5.7-and 8.9-kb genomes and the 110-and 125-kDa polyproteins had been identified in studies on the natural SKVs, the present results not only demonstrate the transforming activity of these individual SKVs but also suggest mechanisms for their generation.

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The nucleotide sequence of a biologically active v-ski gene from a cloned proviral segment shows that ski is a 1,312-base sequence embedded in the p19 region of the avian leukosis virus gag gene. The v-ski sequence contains a single open translational reading frame that encodes a polypeptide with a molecular mass of 49,000 daltons. The predicted amino acid sequence includes nuclear localization motifs that have been identified in other nuclear oncoproteins. It also contains a proline-rich region and a set of cysteine and histidine residues that could constitute a metal-binding domain. Two regions of the amino acid sequences of v-ski and v-myc are related, and the two proteins exhibit similar distributions of hydrophobic and hydrophilic amino acids. Cloned segments of the chicken c-ski proto-oncogene totaling 65 kilobases have been analyzed, and regions related to v-ski have been sequenced. The results indicate that v-ski is derived from at least five coding exons of c-ski, that it is correctly spliced, and that it is missing c-ski coding sequences at both its 5' and 3' ends. The c-ski and avian leukosis virus sequences that overlap the 5' virus/v-ski junction in Sloan-Kettering virus contain an 18-of-20-base sequence match that presumably played a role in the transduction of ski by facilitating virus/c-ski recombination.