Tumor therapy with targeted atomic nanogenerators.

A single, high linear energy transfer alpha particle can kill a target cell. We have developed methods to target molecular-sized generators of alpha-emitting isotope cascades to the inside of cancer cells using actinium-225 coupled to internalizing monoclonal antibodies. In vitro, these constructs specifically killed leukemia, lymphoma, breast, ovarian, neuroblastoma, and prostate cancer cells at becquerel (picocurie) levels. Injection of single doses of the constructs at kilobecquerel (nanocurie) levels into mice bearing solid prostate carcinoma or disseminated human lymphoma induced tumor regression and prolonged survival, without toxicity, in a substantial fraction of animals. Nanogenerators targeting a wide variety of cancers may be possible.

New lines of host defense: inhibition of Ty1 retrotransposition by Fus3p and NER/TFIIH.

The genomes of all organisms examined contain transposons whose uncontrolled movement threatens genome function. Fortunately, host cells have evolved defense mechanisms to minimize the level of transposition. In this review we discuss recent work showing that proteins involved in signal transduction and RNA transcription/DNA repair inhibit Ty1 retrotransposition in the yeast Saccharomyces cerevisiae. On the basis of these examples, we hypothesize that the level of Ty1 retrotransposition may be modulated in response to environmental stress signals that affect cellular differentiation and DNA repair.

Posttranslational control of Ty1 retrotransposition occurs at the level of protein processing.

High-level expression of a transpositionally competent Ty1 element fused to the inducible GAL1 promoter on a 2 microns plasmid (pGTy1) overcomes transpositional dormancy in Saccharomyces cerevisiae. To investigate the mechanisms controlling the rate of Ty1 retrotransposition, we quantitated transposition and Ty1 gene products in cells induced and uninduced for expression of pGTy1. The increase in Ty1 transposition was 45- to 125-fold greater than the increase in Ty1 RNA effected by pGTy1 induction. Translational efficiency of Ty1 RNA was not altered in transposition-induced cells, since p190TYA1-TYB1 protein synthesis increased in proportion to steady-state Ty1 RNA levels. Therefore, expression of a pGTy1 element increases the efficiency of Ty1 transposition at a posttranslational level. Galactose induction of pGTy1 enhanced TYA1 protein processing and allowed detection of processed TYB1 proteins, which are normally present at very low levels in uninduced cells. When the ability of genomic Ty1 elements to complement defined mutations in HIS3-marked pGTy1 elements was examined, mutations in the protease domain or certain mutations in the integrase domain failed to be complemented, but mutations in the reverse transcriptase domain were partially complemented by genomic Ty1 elements. Therefore, the activity of Ty1 elements in yeast cells may be limited by the availability of Ty1 protease and possibly integrase. These results suggest that Ty1 transposition is regulated at the level of protein processing and that this regulation is overcome by expression of a pGTy1 element.

Transpositional competence and transcription of endogenous Ty elements in Saccharomyces cerevisiae: implications for regulation of transposition.

Transposition of Ty elements in the yeast Saccharomyces cerevisiae occurs through an RNA intermediate. Although Ty RNA accounts for 5 to 10% of the total polyadenylated RNA in a haploid cell, the transposition frequency is only 10(-7) to 10(-8) per gene. To determine whether Ty elements native to the yeast genome are transpositionally competent, two elements were fused to the GAL1 promoter and tested for their ability to transpose. These native elements, Ty1-588 and Ty2-117, transposed at high levels when the GAL1 promoter was induced. Three Ty's identified as spontaneous transpositions in specific target genes were also tested. Of these three, Ty2-917 and the previously characterized element Ty1-H3 were shown to be transpositionally competent. The third element, Ty1-H1, was transposition defective. In addition, we marked the chromosomal copy of Ty1-588 with the NEO gene and demonstrated that Ty1-588NEO was actively transcribed in yeast cells. Ty1-588NEO transcription was regulated by the SPT3 and MAT loci in the same manner as that observed for Ty's collectively. These results indicate that the yeast genome contains functional Ty elements. The presence of a transpositionally competent, actively transcribed element suggests that regulation of Ty transposition occurs at a posttranscriptional level.

The Sgs1 helicase of Saccharomyces cerevisiae inhibits retrotransposition of Ty1 multimeric arrays.

Ty1 retrotransposons in the yeast Saccharomyces cerevisiae are maintained in a genetically competent but transpositionally dormant state. When located in the ribosomal DNA (rDNA) locus, Ty1 elements are transcriptionally silenced by the specialized heterochromatin that inhibits rDNA repeat recombination. In addition, transposition of all Ty1 elements is repressed at multiple posttranscriptional levels. Here, we demonstrate that Sgs1, a RecQ helicase required for genome stability, inhibits the mobility of Ty1 elements by a posttranslational mechanism. Using an assay for the mobility of Ty1 cDNA via integration or homologous recombination, we found that the mobility of both euchromatic and rDNA-Ty1 elements was increased 32- to 79-fold in sgs1Delta mutants. Increased Ty1 mobility was not due to derepression of silent rDNA-Ty1 elements, since deletion of SGS1 reduced the mitotic stability of rDNA-Ty1 elements but did not stimulate their transcription. Furthermore, deletion of SGS1 did not significantly increase the levels of total Ty1 RNA, protein, or cDNA and did not alter the level or specificity of Ty1 integration. Instead, Ty1 cDNA molecules recombined at a high frequency in sgs1Delta mutants, resulting in transposition of heterogeneous Ty1 multimers. Formation of Ty1 multimers required the homologous recombination protein Rad52 but did not involve recombination between Ty1 cDNA and genomic Ty1 elements. Therefore, Ty1 multimers that transpose at a high frequency in sgs1Delta mutants are formed by intermolecular recombination between extrachromosomal Ty1 cDNA molecules before or during integration. Our data provide the first evidence that the host cell promotes retrotransposition of monomeric Ty1 elements by repressing cDNA recombination.

Posttranslational regulation of Ty1 retrotransposition by mitogen-activated protein kinase Fus3.

Ty1 retrotransposons in Saccharomyces cerevisiae are maintained in a state of transpositional dormancy. We isolated a mutation, rtt100-1, that increases the transposition of genomic Ty1 elements 18- to 56-fold but has little effect on the transposition of related Ty2 elements. rtt100-1 was shown to be a null allele of the FUS3 gene, which encodes a haploid-specific mitogen-activated protein kinase. In fus3 mutants, the levels of Ty1 RNA, protein synthesis, and proteolytic processing were not altered relative to those in FUS3 strains but steady-state levels of TyA, integrase, and reverse transcriptase proteins and Ty1 cDNA were all increased. These findings suggest that Fus3 suppresses Ty1 transposition by destabilizing viruslike particle-associated proteins. The Fus3 kinase is activated through the mating-pheromone response pathway by phosphorylation at basal levels in naive cells and at enhanced levels in pheromone-treated cells. We demonstrate that suppression of Ty1 transposition in naive cells requires basal levels of Fus3 activation. Substitution of conserved amino acids required for activation of Fus3 derepressed Ty1 transposition. Moreover, epistasis analyses revealed that components of the pheromone response pathway that act upstream of Fus3, including Ste4, Ste5, Ste7, and Ste11, are required for the posttranslational suppression of Ty1 transposition by Fus3. The regulation of Ty1 transposition by Fus3 provides a haploid-specific mechanism through which environmental signals can modulate the levels of retrotransposition.

An alpha-particle emitting antibody ([213Bi]J591) for radioimmunotherapy of prostate cancer.

A novel alpha-particle emitting monoclonal antibody construct targeting the external domain of prostate-specific membrane antigen (PSMA) was prepared and evaluated in vitro and in vivo. The chelating agent, N-[2-amino-3-(p-isothiocyanatophen-yl)propyl]-trans-cyclohexane-1, 2-diamine-N,N',N',N'',N''-pentaacetic acid, was appended to J591 monoclonal antibody to stably bind the 213Bi radiometal ion. Bismuth-213 is a short-lived (t 1/2 = 46 min) radionuclide that emits high energy alpha-particles with an effective range of 0.07-0.10 mm that are ideally suited to treating single-celled neoplasms and micrometastatic carcinomas. The LNCaP prostate cancer cell line had an estimated 180,000 molecules of PSMA per cell; J591 bound to PSMA with a 3-nM affinity. After binding, the radiolabeled construct-antigen complex was rapidly internalized into the cell, carrying the radiometal inside. [213Bi]J591 was specifically cytotoxic to LNCaP. The LD50 value of [213Bi]J591 was 220 nCi/ml at a specific activity of 6.4 Ci/g. The potency and specificity of [213Bi]J591 directed against LNCaP spheroids, an in vitro model for micrometastatic cancer, also was investigated. [213Bi]J591 effectively stopped growth of LNCaP spheroids relative to an equivalent dose of the irrelevant control [213Bi]HuM195 or unlabeled J591. Cytotoxicity experiments in vivo were carried out in an athymic nude mouse model with an i.m. xenograft of LNCaP cells. [213Bi]J591 was able to significantly improve (P < 0.0031) median tumor-free survival (54 days) in these experiments relative to treatment with irrelevant control [213Bi]HuM195 (33 days), or no treatment (31 days). Prostate-specific antigen (PSA) was also specifically reduced in treated animals. At day 51, mean PSA values were 104 ng/ml +/- 54 ng/ml (n = 4, untreated animals), 66 ng/ml +/- 16 ng/ml (n = 6, animals treated with [213Bi]HuM195), and 28 ng/ml +/- 22 ng/ml (n = 6, animals treated with [213Bi]J591). The reduction of PSA levels in mice treated with [213Bi]J591 relative to mice treated with [213Bi]HuM195 and untreated control animals was significant with P < 0.007 and P < 0.0136, respectively. In conclusion, a novel [213Bi]-radiolabeled J591 has been constructed that selectively delivers alpha-particles to prostate cancer cells for potent and specific killing in vitro and in vivo.

Heterogeneous functional Ty1 elements are abundant in the Saccharomyces cerevisiae genome.

Despite the abundance of Ty1 RNA in Saccharomyces cerevisiae, Ty1 retrotransposition is a rare event. To determine whether transpositional dormancy is the result of defective Ty1 elements, functional and defective alleles of the retrotransposon in the yeast genome were quantitated. Genomic Ty1 elements were isolated by gap repair-mediated recombination of pGTy1-H3(delta 475-3944) HIS3, a multicopy plasmid containing a GAL1/Ty1-H3 fusion element lacking most of the gag domain (TYA) and the protease (PR) and integrase (IN) domains. Of 39 independent gap repaired pGTyHIS3 elements isolated, 29 (74%) transposed at high levels following galactose induction. The presence of restriction site polymorphisms within the gap repaired region of the 29 functional pGTyHIS3 elements indicated that they were derived from at least eight different genomic Ty1 elements and one Ty2 element. Of the 10 defective pGTyHIS3 elements, one was a partial gap repair event while the other nine were derived from at least six different genomic Ty1 elements. These results suggest that most genomic Ty1 elements encode functional TYA, PR and IN proteins. To understand how functional Ty1 elements are regulated, we tested the hypothesis that a TYB protein associates preferentially in cis with the RNA template that encodes it, thereby promoting transposition of its own element. A genomic Ty1 mhis3AI element containing either an in-frame insertion in PR or a deletion in TYB transposed at the same rate as a wild-type Ty1mhis3AI allele, indicating that TYB proteins act efficiently in trans. This result suggests in principle that defective genomic Ty1 elements could encode trans-acting repressors of transposition; however, expression of only one of the nine defective pGTy1 isolates had a negative effect on genomic Ty1 mhis3AI element transposition in trans, and this effect was modest. Therefore, the few defective Ty1 elements in the genome are not responsible for transpositional dormancy.

A novel Ty1-mediated fragmentation method for native and artificial yeast chromosomes reveals that the mouse steel gene is a hotspot for Ty1 integration.

We have developed a powerful new tool for the physical analysis of genomes called Ty1-mediated chromosomal fragmentation and have used the method to map 24-retrotransposon insertions into two different mouse-derived yeast artificial chromosomes (YACs). Expression of a plasmid-encoded GAL1:Ty1 fusion element marked with the retrotransposition indicator gene, ade2AI, resulted in a high fraction of cells that sustained a single Ty1 insertion marked with ADE2. Strains in which Ty1ADE2 inserted into a YAC were identified by cosegregation of the ADE2 gene with the URA3-marked YAC. Ty1ADE2 elements also carried a site for the endonuclease I-DmoI, which we demonstrate is not present anywhere in the yeast genome. Consequently, I-DmoI cleaved a single chromosome or YAC at the unique site of Ty1ADE2 insertion, allowing rapid mapping of integration events. Our analyses showed that the frequency of Ty1ADE2 integration into YACs is equivalent to or higher than that expected based on random insertion. Remarkably, the 50-kb transcription unit of the mouse Steel locus was shown to be a highly significant hotspot for Ty1 integration. The accessibility of mammalian transcription units to Ty1 insertion stands in contrast to that of yeast transcription units.

Multiple regulators of Ty1 transposition in Saccharomyces cerevisiae have conserved roles in genome maintenance.

Most Ty1 retrotransposons in the genome of Saccharomyces cerevisiae are transpositionally competent but rarely transpose. We screened yeast mutagenized by insertion of the mTn3-lacZ/LEU2 transposon for mutations that result in elevated Ty1 cDNA-mediated mobility, which occurs by cDNA integration or recombination. Here, we describe the characterization of mTn3 insertions in 21 RTT (regulation of Ty1 transposition) genes that result in 5- to 111-fold increases in Ty1 mobility. These 21 RTT genes are EST2, RRM3, NUT2, RAD57, RRD2, RAD50, SGS1, TEL1, SAE2, MED1, MRE11, SCH9, KAP122, and 8 previously uncharacterized genes. Disruption of RTT genes did not significantly increase Ty1 RNA levels but did enhance Ty1 cDNA levels, suggesting that most RTT gene products act at a step after mRNA accumulation but before cDNA integration. The rtt mutations had widely varying effects on integration of Ty1 at preferred target sites. Mutations in RTT101 and NUT2 dramatically stimulated Ty1 integration upstream of tRNA genes. In contrast, a mutation in RRM3 increased Ty1 mobility >100-fold without increasing integration upstream of tRNA genes. The regulation of Ty1 transposition by components of fundamental pathways required for genome maintenance suggests that Ty1 and yeast have coevolved to link transpositional dormancy to the integrity of the genome.

Posttranslational inhibition of Ty1 retrotransposition by nucleotide excision repair/transcription factor TFIIH subunits Ssl2p and Rad3p.

rtt4-1 (regulator of Ty transposition) is a cellular mutation that permits a high level of spontaneous Ty1 retrotransposition in Saccharomyces cerevisiae. The RTT4 gene is allelic with SSL2 (RAD25), which encodes a DNA helicase present in basal transcription (TFIIH) and nucleotide excision repair (NER) complexes. The ssl2-rtt (rtt4-1) mutation stimulates Ty1 retrotransposition, but does not alter Ty1 target site preferences, or increase cDNA or mitotic recombination. In addition to ssl2-rtt, the ssl2-dead and SSL2-1 mutations stimulate Ty1 transposition without altering the level of Ty1 RNA or proteins. However, the level of Ty1 cDNA markedly increases in the ssl2 mutants. Like SSL2, certain mutations in another NER/TFIIH DNA helicase encoded by RAD3 stimulate Ty1 transposition. Although Ssl2p and Rad3p are required for NER, inhibition of Ty1 transposition is independent of Ssl2p and Rad3p NER functions. Our work suggests that NER/TFIIH subunits antagonize Ty1 transposition posttranslationally by inhibiting reverse transcription or destabilizing Ty1 cDNA.

Radioimmunotherapy for model B cell malignancies using 90Y-labeled anti-CD19 and anti-CD20 monoclonal antibodies.

In recent years, radioimmunotherapy (RIT) with beta(-) particle emitting radionuclides targeting the CD20 antigen on B cells in the treatment of non-Hodgkin's lymphoma has provided the most compelling human clinical data for the success of RIT. CD19, like CD20, is an antigen expressed on the surface of cells of the B lineage, and CD19 may provide an alternative target for radioimmunotherapy of B cell neoplasms. CD19 has been largely overlooked as a target for conventional 131I RIT, because the antigen rapidly internalizes upon binding of antibody, resulting in catabolism and significant release of 131I. Such modulation may be an advantage to RIT with radiometals such as 90Y, 177Lu, 213Bi and 225Ac. Herein, we have compared beta(-) particle RIT with antibodies targeting either CD19 or CD20. The anti-CD19 and anti-CD20 antibodies, B4 or C2B8, respectively, were appended with the SCN-CHX-A''-DTPA bifunctional chelating agent and labeled with 90Y. In the tumor model used, there were three times as many CD20 target sites on lymphoma cells as compared to CD19 sites (62000 vs 20000 binding sites, respectively). We compared the efficacy of the 90Y-labeled antibodies to reduce lymphoma in a nude mouse xenograft solid tumor model, after measurable lymphoma appeared. Reduction in tumor size began at day 3 in all three 90Y-treated groups, but tumor began to recur in many animals 9 days after the treatments. There was one cure in each specific treatment group. In contrast, the tumor in the two control groups showed no regression. There was a significant prolongation of median survival time from xenograft (P < 0.0001) in all the 90Y-labeled antibody construct-treated groups (32 days for 0.15 mCi 90Y-B4; 26 days for 0.20 mCi 90Y-C2B8, and 23 days for 0.15 mCi 90Y-C2B8) in comparison to the two control groups (11 days for 0.02 mg of C2B8 and 9 days for untreated growth controls). Specificity of the radioimmunotherapy was also shown. In conclusion, 90Y-labeled anti-CD19 antibody has efficacy comparable to 90Y-labeled anti-CD20 antibody in the treatment of mice bearing human lymphoma xenografts. These data suggest that CD19-targeted RIT merits further study.

Impact of the high tyrosine fraction in complementarity determining regions: measured and predicted effects of radioiodination on IgG immunoreactivity.

Although iodine-131 is the most widely used radionuclide for radioimmunotherapy, direct radiolabeling methods yield decreased immunoreactivity of the antibody as a function of increased iodine incorporation. We have studied the amino acid sequences of a therapeutic IgG (HuM195), and in particular its complementarity determining regions (CDR), in order to correlate the iodination of tyrosine residues in the antigen binding site with changes in immunoreactivity. The CDR contained an overabundance of tyrosines relative to an expected random distribution of amino acids. In contrast, lysine residues that can be used for ligand attachment were evenly distributed throughout the IgG. HuM195 was first trace labeled with 111In and then labeled with stable 127I at various specific activities. The immunoreactivity of each product was determined using the 111In tracer. The immunoreactivity measured after varying levels of iodination fit a theoretical curve that was generated based on the assumption that a single iodine incorporation anywhere on a tyrosine residue in a CDR destroys the immunoreactivity of the antibody. Similar theoretical curves for antibody fragments (Fab, Fv) suggest an even faster decrease in immunoreactivity with increasing iodination. A review of the sequences of other therapeutic IgG shows that a similar overabundance of tyrosine residues is found in the CDRs. Using enzyme digestion, the distribution of iodine on different parts of the antibody was also studied. The iodinated residues were distributed non uniformly throughout the IgG, with the heavy chain variable region tyrosines having a higher propensity for iodine incorporation than tyrosines in the other regions of the IgG. The common abundance of tyrosine in the CDR of IgG and its correlation with loss of function have important implications for therapeutic use of high specific activity radioiodinated monoclonal antibodies or fragments.

Alpha-emitting bismuth cyclohexylbenzyl DTPA constructs of recombinant humanized anti-CD33 antibodies: pharmacokinetics, bioactivity, toxicity and chemistry.

UNLABELLED: The alpha-particle-emitting radionuclides have several physical characteristics that make them attractive candidates for radioimmunotherapy: (a) high linear energy transfer; (b) short path lengths (50-80 microm); and (c) limited ability of cells to repair damage to DNA. This article describes the pharmacokinetic, bioactivity, toxicity and chemical characteristics of alpha-particle-emitting, 213Bi and 212Bi radiometal conjugated HuM195 (anti-CD33) constructs. Conjugation of HuM195 to SCN-CHX-A-DTPA resulted in the attachment of up to 10 chelating ligand molecules per antibody. RESULTS: Radiolabeling efficiency of the CHX-A-DTPA-HuM195 construct with 213Bi was 78%+/-10% (n = 46) after 10 min at specific activities of up to 1110 MBq/mg. The immunoreactivity of the 213Bi-labeled CHX-A-DTPA-HuM195 construct was 84%+/-10% (n = 28) and was independent of the specific activity. The bismuth-labeled CHX-A-DTPA-HuM195 construct was rapidly internalized into the cell in a time-dependent manner ranging from 50% at 1 h to 65% at 24 h. 205Bi/206Bi-labeled constructs were stable for at least 2 d in vitro in the presence of human serum at 37 degrees C. After injection into mice, there was no uptake or loss of bismuth to mouse tissues, which do not express CD33, or to the kidney, which has avidity for free bismuth. Mice injected intraperitoneally with doses of (213Bi)CHX-A-DTPA-HuM1 95 ranging from 18.5 to 740 MBq/kg showed no toxicity, but at 2590 MBq/kg, two of the three mice died within 2 wk and a third mouse showed significant reductions in white blood cell counts. Mice injected intravenously with doses of (213Bi)CHX-A-DTPA-HuM195 up to 370 MBq/kg exhibited little toxicity, but 666 MBq/kg was above the MTD for mice. Leukemia cell killing in vitro with bismuth-labeled HuM1 95 showed dose- and specific activity-dependent killing of CD33+ HL60 cells; approximately 50% killing was observed when two bismuth atoms (50 fM radiolabeled antibody) were initially bound onto the target cell surface. CONCLUSION: Alpha-emitting antibodies are among the most potent cytotoxic agents known, yet are specific and appear safe in vivo. The physical and biochemical characteristics of the 213Bi isotope and its generation, as well as the biochemistry of the 213Bi-labeled CHX-A-DTPA-HuM195 construct, make it possible to use the constructs safely and feasibly in humans at therapeutic levels.

A rapid, single vessel method for preparation of clinical grade ligand conjugated monoclonal antibodies.

A rapid, single vessel method for the preparation of clinical grade chelate conjugated monoclonal antibodies has been developed. By use of an Amicon concentrator with reservoir, each of the steps necessary for the preparation of the conjugated drug may be performed in a single vessel. Advantages include reduced risk of metal, pyrogen and bacterial contamination; buffer exchanges are achieved rapidly and efficiently using a continuous dilution method. The radiolabeling efficiency, the radiochemical purity, the total immunoreactivity and the affinity of the final product have been evaluated in the production of CHXA-DTPA-chelate conjugated HuM195. The characteristics compare favorably to those achieved using our conventional synthetic methods.

Regulation of retrotransposition in Saccharomyces cerevisiae.

Retrotransposons are a widely distributed group of eukaryotic mobile genetic elements that transpose through an RNA intermediate. The element Ty (Transposon yeast), found in the yeast Saccharomyces cerevisiae, is a model system for the study of retrotransposons because of the experimental tools that exist to manipulate and detect transposition. Ty transposition can be elevated to levels exceeding one transposition event per cell when an element is expressed from an inducible yeast promoter. In addition, individual genomic Ty elements can be tagged with a retrotransposition indicator gene that allows transposition events occurring at a rate of 10(-5) to 10(-7) per element per cell division to be detected phenotypically. These systems are being used to elucidate the mechanism of Ty transposition and clarify how Ty transposition is controlled.

Single-step selection for Ty1 element retrotransposition.

The yeast retrotransposon Ty1 has been tagged with a reporter gene that allows selection of RNA-mediated transposition events and is applicable to the study of retroelements in other organisms. The reporter gene is a yeast HIS3 gene interrupted by an artificial intron (AI) in the antisense orientation. The HIS3AI sequences were inserted into a Ty1 element such that the intron is on the sense strand of the Ty1 element; therefore, splicing and retrotransposition of marked Ty1 transcripts can give rise to His+ cells. Fusion of the Ty1-H3mHIS3AI element to the inducible GAL1 promoter resulted in a high frequency of histidine prototrophs upon galactose induction. Moreover, spontaneous His+ revertants derived from strains containing genomic TymHIS3AI elements are a result of retrotransposition. By using this assay, we estimated the Ty1 transposition rate to be between 3 x 10(-7) and 1 x 10(-5) transpositions per Ty1 element per generation. Variations in the transposition rate of individual Ty1 elements are correlated with the relative abundance of their transcripts.

Fus3 controls Ty1 transpositional dormancy through the invasive growth MAPK pathway.

Fus3, the mitogen-activated protein kinase (MAPK) of the mating pheromone response pathway, inhibits a post-translational step of Ty1 retrotransposition. Fus3 also inhibits haploid invasive growth by blocking cross-activation of invasive growth gene expression by the pheromone response signal cascade. Here, we show that Fus3 kinase activity and dosage co-ordinately regulate Ty1 transposition and invasive growth. A chromosomal copy of the kinase-defective fus3-K42R allele fails to inhibit either Ty1 transposition or invasive growth. When overexpressed, kinase-defective Fus3 weakly inhibits both Ty1 transposition and invasive growth, but is much less inhibitory than wild-type Fus3 expressed at the same level. Moreover, increasing the dosage of wild-type Fus3 intensifies the inhibition of both Ty1 transposition and invasive growth. To demonstrate that Fus3 regulates Ty1 transposition via its negative regulation of the invasive growth pathway, we show by epistatic analysis that the invasive growth pathway transcription factors Ste12 and Tec1 are both required for Fus3-mediated inhibition of Ty1 transposition. When haploid invasive growth is stimulated by high-copy expression of TEC1, by expression of the dominant hypermorphic allele STE11-4 or by deletion of HOG1, Ty1 transposition is concomitantly activated. In summary, these results demonstrate that the haploid invasive growth pathway activates Ty1 transposition at both transcriptional and post-transcriptional levels and that Fus3 inhibits Ty1 transposition by inhibiting the invasive growth pathway.

Ty RNA levels determine the spectrum of retrotransposition events that activate gene expression in Saccharomyces cerevisiae.

To learn more about the variety of Ty elements capable of activating gene expression, we characterized 206 spontaneous Ty transpositions that activate the promoterless gene his3 delta 4. Most of the Ty elements appear to be full-length, although a few deleted elements were recovered. Over 95% of the insertions belong to the Ty1 family, and the rest are Ty2 elements. The excessive number of Ty1 transpositions was unexpected because there are only 2-fold more Ty1 than Ty2 elements in the yeast strains used in the selection. However, there is 20-fold more Ty1 than Ty2 RNA present in these yeast strains. This difference in RNA level explains the greater number of Ty1 verses Ty2 transpositions at his3 delta 4, because Ty elements transpose through an RNA intermediate. A similar association between the Ty transcript level and transpositional activation of his3 delta 4 is obtained in cells expressing GAL1-promoted Ty2-H556 or Ty2-917 elements, but only if the element does not contain a marker. Genetically marked Ty2-H556NEO and -917NEO elements transpose into and activate his3 delta 4 with the same efficiency as the previously characterized Ty1-H3NEO element, but are underrepresented relative to the levels of TyNEO transcript. We also found that chromosomal Ty transcripts are even more abundant than previously estimated and comprise about 1% of total cellular RNA.