Faculty Bibliography

We have found reverse transcriptase activity and virus-like particles only in yeast cells that contain a galactose-promoted Ty element induced on galactose. The cofractionation of reverse transcriptase, genomic-length Ty RNA, and a Ty-specified protein antigen in a particulate fraction and the ability of this complex to synthesize specifically a product that is homologous to the entire Ty suggest that reverse transcription of Ty RNA takes place in the particle. The absence of appreciable levels of reverse transcriptase and particles in uninduced cells despite the presence of at least 35 copies of chromosomal Ty elements suggest that some of these elements may be defective. The numerous virus-like particles visible in thin sections of Ty transposition-induced cells appear not to be infectious. These particles resemble the intracisternal A-type particles of the mouse and copia particles of Drosophila. The results support the idea that Ty elements and retroviruses share a common origin.

We have followed Ty transposition with a donor Ty element, TyH3, whose expression is under the control of the GAL1 promoter. Sequence analysis reveals dramatic structural differences in TyH3 before and after transposition. If the donor TyH3 is marked with an intron-containing fragment, the intron is correctly spliced out of the Ty during transposition, suggesting that the Ty RNA is the intermediate for transposition. Furthermore, the pattern of sequence inheritance in progeny Ty insertions derived from the marked Ty follows the predictions of the model of retroviral reverse transcription. Comparison of marked Ty elements before and after movement shows that transposition is highly mutagenic to the Ty element. These results demonstrate that during transposition, Ty sequence information flows from DNA to RNA to DNA.

We describe a detailed deletion analysis of the anchoring domain of a model membrane protein. Removal of the 23 contiguous uncharged amino acids from the carboxy terminus of the bacteriophage fl gene III protein (pIII) converts it from an integral membrane protein to a secreted periplasmic form. Deletions that remove six or fewer residues of the hydrophobic core result in no diminution of the protein's capacity to anchor in the membrane. Longer deletions into this hydrophobic domain gradually destablize the protein-membrane association. pIII derivatives with over half of the hydrophobic core deleted retain substantial residual anchor function. The basic residues, arginine and lysine, which provide a carboxy-terminal boundary for this domain, can be deleted without loss of anchoring capacity.

Mutations at the URA3 locus of Saccharomyces cerevisiae can be obtained by a positive selection. Wild-type strains of yeast (or ura3 mutant strains containing a plasmid-borne URA3+ gene) are unable to grow on medium containing the pyrimidine analog 5-fluoro-orotic acid, whereas ura3- mutants grow normally. This selection, based on the loss of orotidine-5'-phosphate decarboxylase activity seems applicable to a variety of eucaryotic and procaryotic cells.

The histidine-rich protein (HRP) of the avian malaria parasite Plasmodium lophurae contains 70% histidine. It is found in dense cytoplasmic granules and during the erythrocytic cycle it accumulates to represent 10% of the dry weight of the parasite. In the present work the HRP mRNA was studied by in vitro translation and by the use of a polyhistidine oligonucleotide probe. The HRP mRNA contains 2,000-2,100 nucleotides encoding a protein with an apparent molecular weight of 50,000. In addition a HRP of molecular weight 35,000-40,000 is also produced in vitro, probably as a result of proteolytic cleavage of the molecular weight 50,000 polypeptide which corresponds to in vivo labeled and purified HRP. The HRP represents a much larger proportion of the in vitro products synthesized in the homologous cell-free system compared to the rabbit reticulocyte system, and it reflects more closely the pattern of protein synthesis seen in vivo. In addition, HRP mRNA is more abundant in polysomes isolated from young parasites than in polysomes from mature schizonts. These results indicate that the HRP accumulates as a result of amplified translation of its mRNA at certain stages of its erythrocytic cycle.

A Staphylococcus aureus plasmid derivative, pFB9, coding for erythromycin and chloramphenicol resistance was cloned into the filamentous Escherichia coli phage f1. Recombinant phage-plasmid hybrids, designated plasmids, were isolated from E. coli and purified by transformation into Streptococcus pneumoniae. Single-stranded DNA was prepared from E. coli cells infected with two different plasmids, fBB101 and fBB103. Introduction of fully or partially single-stranded DNA into Streptococcus pneumoniae was studied, using a recipient strain containing an inducible resident plasmid. Such a strain could rescue the donor DNA marker. Under these marker rescue conditions, single-stranded fBB101 DNA gave a 1% transformation frequency, whereas the double-stranded form gave about a 31% frequency. Transformation of single-stranded fBB101 DNA was inhibited by competing double-stranded DNA and vice versa, indicating that single-stranded DNA interacts with the pneumococcus via the same binding site as used by double-stranded DNA. Heteroduplexed DNA containing the marker within a 70- or 800-base single-stranded region showed only slightly greater transforming activity than pure single-stranded DNA. In the absence of marker rescue, both strands of such imperfectly heteroduplexed DNA demonstrated transforming activity. Pure single-stranded DNA demonstrated low but significant transforming activity into a plasmid-free recipient pneumococcus.

Gene III protein of bacteriophage f1 is inserted into the host cell membrane where it is assembled into phage particles. A truncated form of gene III protein, encoded by a recombinant plasmid and lacking the carboxyl terminus, does not remain in the membrane but instead appears to slip through it. Fusion of a hydrophobic "membrane anchor" from another membrane protein, the gene VIII protein, to the truncated gene III protein (by manipulation of the recombinant plasmid) restores membrane anchoring. A model for the relationship of gene III protein with the Escherichia coli membrane is discussed.

Derivatives of filamentous phage, f1, fd, and M13, useful as cloning vectors are listed, and procedures for their use are reviewed. Methods for growing phage, preparing single- and double-stranded DNA, and cloning are given in the "cook-book" form. These procedures minimize the practical problem often associated with filamentous-phage cloning, i.e., deletion of inserts.