Faculty Bibliography

Mutations in the SPT10 and SPT21 genes were originally isolated as suppressors of Ty and LTR (delta) insertion mutations in Saccharomyces cerevisiae, and the genes were shown to be required for normal transcription at a number of loci in yeast. Now we have cloned, sequenced, mapped and mutagenized SPT10 and SPT21. Since the spt10 mutation used to clone SPT10 resulted in very poor transformation efficiency, a novel method making use of the kar1-1 mutation was used. Neither SPT gene is essential for growth, and constructed null alleles cause phenotypes similar to those caused by spontaneous mutations in the genes. spt10 null alleles are strong suppressor mutations and cause extremely slow growth. Certain spt10 spontaneous alleles are good suppressors but have a normal growth rate, suggesting that the SPT10 protein may have two distinct functions. An amino acid sequence motif that is similar to the Zn-finger motif was found in SPT10. Mutation of the second Cys residue in this motif resulted in loss of complementation of the suppression phenotype but a normal growth rate. Thus, this motif may reside in a part of the SPT10 protein that is important for transcriptional regulation but not for normal growth. Both the SPT10 and SPT21 proteins are relatively tolerant of large deletions; in both cases deletions of the C-terminus resulted in at least partially functional proteins; also, a large internal deletion in SPT21 was phenotypically wild type.

Yeast RNA lariat debranching enzyme has been purified to near homogeneity using a bacterial overproducer of the enzyme. The enzyme is capable of digesting a variety of branched nucleic acid substrates, including group II intron lariats, multicopy single-stranded DNAs (msDNAs), and a variety of synthetic branched RNAs. A trinucleotide release assay using radiolabeled msDNA substrates was developed and used to determine the basic biochemical parameters for the enzyme. The debranching enzyme shows a strong preference for purines at the 2'-position in both msDNA and synthetic branched RNA substrates, in accord with the structure of its native substrate, which always has a 2'-G residue. The use of small synthetic branched RNA substrates will allow systematic mechanistic and structural studies of this unique enzyme.

A plasmid bearing a transpositionally functional GAL1::Ty1 fusion was mutagenized by insertion of four or five codons semirandomly throughout the plasmid. This collection of mutant plasmids was introduced into yeast cells and studied with regard to the properties of the mutant Ty1-encoded proteins and the transposition phenotypes observed. All of the transposition-inactivating mutations were previously found to be recessive with the exception of a single mutation in TYA. In this mutant, TYA protein of normal abundance is produced, but the virus-like particles containing this protein are unstable and have aberrant behavior. The effects of mutations in noncoding regions, as well as the capsid protein coding region TYA, and the regions encoding the protease, integrase and reverse transcriptase proteins are described. Effects on gene expression, types of proteins produced, proteolysis of precursor proteins, virus-like particle structure, and biochemical activities of the encoded proteins are summarized. In addition, we show that one of the mutations in the 3' LTR represents a new nonessential site into which foreign marker DNA can be inserted without compromising transposition.

L1 elements constitute a highly repetitive human DNA family (50,000 to 100,000 copies) lacking long terminal repeats and ending in a poly(A) tail. Some L1 elements are capable of retrotransposition in the human genome (Kazazian, H. H., Jr., C. Wong, H. Youssoufian, A. F. Scott, D. G. Phillips, and S.E. Antonarakis, Nature (London) 332:164-166, 1988). Although most are 5' truncated, a consensus sequence of complete L1 elements is 6 kb long and contains two open reading frames (ORFs) (Scott, A. F., B. J. Schmeckpeper, M. Abdelrazik, C. T. Comey, B. O'Hara, J. P. Rossiter, T. Cooley, P. Health, K. D. Smith, and L. Margolet, Genomics 1:113-125, 1987). The protein encoded by ORF2 has reverse transcriptase (RT) activity in vitro (Mathias, S. L., A. F. Scott, H. H. Kazazian, Jr., J. D. Boeke, and A. Gabriel, Science 254:1808-1810, 1991). Because L1 elements are so numerous, efficient methods for identifying active copies are required. We have developed a simple in vivo assay for the activity of L1 RT based on the system developed by Derr et al. (Derr, L. K., J. N. Strathern, and D. J. Garfinkel, Cell 67:355-364, 1991) for yeast HIS3 pseudogene formation. L1 ORF2 displays an in vivo RT activity similar to that of yeast Ty1 RT in this system and generates pseudogenes with unusual structures. Like the HIS3 pseudogenes whose formation depends on Ty1 RT, the HIS3 pseudogenes generated by L1 RT are joined to Ty1 sequences and often are part of complex arrays of Ty1 elements, multiple HIS3 pseudogenes, and hybrid Ty1/L1 elements. These pseudogenes differ from those previously described in that there are base pairs of unknown origin inserted at several of the junctions. In two of three HIS3 pseudogenes studied, the L1 RT appears to have jumped from the 5' end of a Ty1/L1 transcript to the poly(A) tract of the HIS3 RNA.

Mutations within the TYB gene of Ty1 encoding integrase (IN) as well as alterations in its substrate, a linear DNA molecule, were examined for their effects on in vitro IN activity, using a recently developed physical assay. Five different codon-insertion mutations, two frameshift mutations, and one missense mutation, previously identified as transposition-deficient mutations, were tested. Virus-like particles, the source of IN, from two different protease mutants and a reverse transcriptase mutant exhibited near-normal to normal IN activity. Two frameshift mutations mapping within the phylogenetically variable C-terminal domain of IN resulted in significant in vitro IN activity. In contrast, three mutations within the amino-terminal conserved domain of IN completely abolished IN activity. When the substrate termini were mutated, we found that substrates with as few as 4 bp of Ty1 termini were capable of efficiently generating integration products. Surprisingly, certain substrates that lacked obvious similarity to Ty1 termini were also readily integrated into both linear and circular targets, whereas others were not used as substrates at all. Termini rich in adenosine residues were among the more active substrates; however, certain substrates lacking terminal adenosine residues can form small quantities of integration products, including complete integration reactions.