Thomas H. Eickbush - US grants

The long term objective of this project is to obtain an understanding of the mechanisms by which the activities of disparate genes are temporally controlled. The system I have chosen as a model of such regulation is the production of the chorion (eggshell) by the monolayer of follicle cells surrounding the maturing oocytes of the commericial silkmoth, Bombyx mori. In these cells the synthesis of well over 100 chorion proteins follows a precise temporal pattern corresponding to a program of specific mRNA production. The majority of these chorion proteins are members of large multigene families. At least 50 percent of these genes have been localized to continuous segments of DNA 370 kb in length. The remaining chorion genes will be cloned and linked to these segments using modified chromosomal walking procedures. The individual genes encountered in the locus will be classified as to their respective gene family and to their precise period of expression in choriogenesis. Remarkable variability of the DNA level has been detected between different inbred strains of B. mori. A detailed description of this variation will be obtained by additional chromosomal walking in these strains. Any conserved features revealed by this analysis will indicate elements critical to the maintenance of coordinate gene expression. The variable features will suggest the processes by which the gene families can change during evolution. Two possible mechanism for the regulation of chorion gene expression will be tested. Chromatin accessibility will be explored by monitoring developmental changes in DNase sensitivity, state of methylation or association with the nuclear matrix. Regulation by means of promoter structure will be explored by comparative DNA sequence determination of the ca. 250 bp 5' regions separating the divergently oriented chorion gene pairs.

The long-term objective of this project is to obtain an understanding of how an elaborate developmental process is accomplished by the integrated expression of a large number of genes. Failure to integrate the expression of various structural genes is the basis for a variety of human developmental defects. The model system chosen for this study is the production of the chorion (eggshell) by the monolayer of follicle cells surrounding the maturing oocyte of the silkmoth, Bombyx mori. In these cells the synthesis of over 100 chorion proteins follows a precise temporal pattern corresponding to a program of specific mRNA production. One useful property of this system is that all chorion genes are tightly clustered on a small segment of one chromosome. The size of this region is estimated to be 700 kilobases. Recovery of the entire locus on recombinant clones (500 kb of which is already accomplished) along with detailed characterizations of the genes encountered, will provide a rare opportunity to trace the evolution of a complex developmental process, as well as provide the groundwork for studies designed to understand how the locus is coordinately regulated. Our study of gene regulation will involve two major approaches. First, utilizing the synchronous populations of cells that can be dissected from the silkmoth ovary, we will examine the structural changes of the chorion genes within the nucleus prior to and during their expression. These structural changes will be monitored by determining the accessibility of the genes to DNase I digestions. Such experiments address the important question of how genes are marked in the nucleus for subsequent expression. Second, we will determine whether the chorion locus is divided into looped chromosomal domains. Evidence for such loops will be obtained by if segments of the chorion locus are found to be associated with the nuclear matrix, an elaborate protein network within the nucleus. The use of mutations containing breakage points within the chorion locus will be extremely helpful in determining the functional role of such putative attachment sites.

Retrotransposable elements are abundant in all eukaryotic genomes. These elements have undoubtly played a significant role in reshaping these genomes, can account for many insertional mutations and chromosomal rearrangements, and are believed to be the origin of several viruses, most significantly retroviruses. Those retrotransposable elements with long-terminal repeats (LTRs) are well-studied and are believed to integrate into the genome by mechanisms similar to retroviruses. A second class of retrotransposable elements, termed here the non-LTR elements, have more recently been discovered and appear to have a fundamentally different mechanism for integration. The specific aim of this proposal is to study the mechanism of retrotransposition of the non-LTR element, R2. This element inserts specifically in the 28S genes of a wide variety of insects. It is known that the R2 element encodes an endonuclease that requires RNA before it can specifically cleave the 28S gene insertion site. The same protein also has reverse transcriptase activity. The specific integration of this element and the ability to express the entire coding capacity of this element in E. coli makes it ideally suited to test basic features of current models of non-LTR retrotransposition. Comparative studies will be conducted with the R2 element of both Bombyx mori and Drosophila melanogaster. The critical questions to be addressed are (a) whether the R2 protein is capable of specifically binding the R2 RNA transcript, and if so what sequences, (b) the nature of the interaction between the endonuclease with the DNA target site before and after cleavage, and (c) whether the cleaved DNA, which is not complimentary to the R2 element, can serve as a primer for reverse transcription. We will also attempt to obtain complete integration of an R2 element into a ribosomal DNA repeat in vivo by injecting the R2-- transcript/R2 protein complex into tissue culture cells. We will use a highly sensitive (PCR) assay to screen for integration. Finally, to lay the foundation for our long term goal of understanding the developmental regulation of R2 retrotransposition, we will attempt to find in D. melanogaster genetic conditions underwhich R2 elements are transcribed and translated, and whether this correlates with the integration of new R2 copies.

R1 and R2 are sequence-specific retrotransposable elements found inserted in a fraction of the 28S ribosomal RNA genes of most arthropods. The objective of this proposal is to determine whether the remarkable success of these elements is due to their freedom to move from species to species or their ability to remain active in a species lineage over long evolutionary periods. Most experiments will focus on the sequence relationships of R1 and R2 isolated from species of the dipteran genus Drosophila. The 3' half of the elements will be cloned after the polymerase chain reaction amplification and their nucleotide sequences determined. This sequence analysis will be complemented with studies of the distribution of R1 and R2 within different geographical strains of several species using genomic DNA blotting procedures. Experiments will also be conducted with three species of parasitic wasp (genus Nasonia) which contain multiple families of highly divergent R1 elements. %%% The proposed experiments will provide information that is fundamental to an understanding of the stability of mobile elements within genomes, their ability to transfer between species, and their ability to regulate their own abundance.

R1 and R2 are two site-specific non-LTR retrotransposable elements that insert in the 28S ribosomal RNA genes of arthropods. Recent experiments have indicated that despite their remarkable evolutionary stability, R1 and R2 rapidly turnover in a population (i.e new elements are inserted and old elements eliminated from the rDNA loci at a high rate). Thus a major focus of the current proposal is experiments designed to determine the parameters that control the level of R1 and R2 within the rDNA loci of Drosophila. Taking advantage of the diverse array of 5' truncations that frequently result from non-LTR retrotransposition, preliminary experiments have shown that individual R2 insertions and deletions can be scored in the Harwich mutation collection lines of D. melanogaster. The Harwich lines have been maintained as 31 small, separate populations for over 250 generations. The hundreds of insertion/deletion events that can be monitored by simply PCR assays of these 31 lines should enable separate measurements of the frequency of R1 and R2 events on the rDNA loci of the X and Y chromosomes, and determine whether they occur in bursts or at low constitutive rates. Initial attempts will also be made to determine why certain species groups of Drosophila have retained two distinct lineages of R1 elements, while other species groups have retained only one R1 lineage. R1 and R2 turnover and rDNA unit uniformity in two species with multiple R1s will be analyzed to test the hypothesis that lower levels of concerted evolution enable the preservation of multiple families. Larval tissues of Drosophila have reduced levels of R1 and R2 insertions (relative to the adult levels) because rDNA units with these insertions are under-replicated during the formation of the polyploid (usually polytene) tissues of larvae. A second series of experiments is designed to directly study the mechanism responsible for this under-replication. The molecular/genetic tools are now available to mark individual rDNA units or to insert new sequences into these units. Because the retrotransposition and evolution of R1 and R2 are so intimately tied to that of the rRNA genes, these studies should also provide insights, as well as supply new tools, for the study of the evolution, replication and expression of the rRNA genes themselves. Finally, all non-LTR retrotransposons can be divided into a limited number of distinct, ancient lineages. The oldest elements are site-specific and encode proteins similar to R2. A third set of experiments in this proposal is designed to identify additional examples of these original site-specific non-LTR elements, particularly in primitive eukaryotic lineages like Giardia. Information obtained from these studies will provide insights into the origins of the first eukaryotic genomes, and help to evaluate the relationship of non-LTR retrotransposable elements to their closest relatives: telomerase and group II introns.

Transposable elements are abundant components of eukaryotic genomes. Numerous examples now exist to indicate that these elements have played a significant role in determining the size, structure and regulation of these genomes. A growing number of transposable elements, particularly the retrotransposable elements, have been shown to minimize, or control, their effects on the host by becoming specific for defined locations within the genome. R1 and R2 are two site-specific non-LTR retrotransposable elements that insert in the 28S ribosomal RNA genes of arthropods. The specificity and sequence uniformity of these elements has enabled them to serve as a convenient model system. Considerable progress has been made in studies of their distribution, evolution and retrotransposition mechanism, such that R1 and R2 are now among the best characterized retrotransposable elements. Extensive studies on the evolution of these elements suggest that R1 and R2 have been stable, active components of the rDNA loci for the entire history of the Arthropod phylum, a period estimated to be over 600 million years. In this proposal, a varied set of experiments are described with the objective of better understanding the remarkable stability and the origins of these elements.

Eukaryotic genomes are filled with sequences derived by the reverse flow of information from RNA to DNA. Insertion of these reverse transcripts can induce spontaneous genetic disorders and cancer and have over evolutionary time completely reshaped eukaryotic genomes by generating enormous amounts of repetitive DNA. The most active, yet poorly understood, machinery responsible for these insertions is encoded by the non-long terminal repeat (non-LTR) retrotransposable elements One of the best characterized non-LTR elements is the R2 element of arthropods. This element exclusively inserts into the 28S rRNA genes of its host which greatly simplifies its characterization. The single protein encoded by R2 has remarkable specificity for both its DNA target site and RNA template enabling in vitro studies of its retrotransposition mechanism. This mechanism involves simple cleavage of the DNA target and the polymerization of the reverse transcript directly onto the cleaved chromosome, and thus is fundamentally different from the well-described retroviral mechanism of retrotransposition. Instead non-LTR elements are related to and their retrotransposition shares features with telomerase, the enzyme responsible for telomeres. The specific aim of this proposal is to fully characterize the R2 retrotranspostion mechanism as a model for all non-LTR elements. Detailed mutagenesis of the protein and nucleic acid components and characterization of the enzymatic activities of the protein are proposed. These in vitro studies will be complemented with in vivo studies of the R2 integration reaction in Drosophila melanogaster. Purified R2 protein/RNA when infected into D. melanogaster embryos can integrate foreign sequences into the 28S genes. This integration system will enable studies of the transcription and translation of R2 elements within the rDNA units of cell's nucleolus. These combined studies will address general questions about the mechanism of non-LTR retrotransposition, as well as questions concerning transcription regulation of the rRNA genes. Finally, a growing number of non-LTR elements have been characteriz4ed that retain different insertion specificities for unique sites in their host's genome. The enzymatic machinery encoded by these elements appears identical to that of R2. Our long term goal is to isolate and alter the target specific binding component of the integration reaction , which enable us to design systems that we will insert foreign sequences at other unique locations in eukaryotic genomes.

Transposable elements have played a major role in determining the size, structure and expression of eukaryotic genomes. R1 and R2 are two site-specific non-LTR retrotransposable elements that insert in the 28S ribosomal RNA genes of arthropods. The specificity of these elements has enabled them to serve as a convenient model system, such that R1 and R2 are now among the best characterized transposable elements. Taking advantage of the diverse array of 5' truncations that frequently result from non-LTR retrotransposition, preliminary experiments have shown that individual R1 and R2 insertions and deletions can be scored among members of the same population as well as among 19 Harwich mutation collection lines of Drosophila melanogaster. Therefore new copies are continually being inserted into and old elements eliminated from the rDNA loci. In this project, experiments are described to address a) the recombinational forces that bring about the concerted evolution of this locus, b) how R1 and R2 elements are affected by these recombinations, and c) how often and when R1 and R2 retrotranspose. Attempts to answer these questions will focus on the following specific aims. The rDNA loci from one Harwich line will be cloned as a series of overlapping bacterial artificial chromosomes (BACs). While the cloning of rDNA loci are avoided in genome projects, it should be achievable in this project because nearly 80 uniquely marked R1 and R2 insertions have been identified to serve as reference points for the assembly of overlapping clones. Based on this physical map, as well as the 18 examples of how that locus has changed over a period of 350 generations, it can be determined where R1 and R2 insert into the loci and whether they are uniformly removed. Second, by scoring changes in the variable intergenic spacer region between the rRNA genes, a detailed view can be obtained of the recombinational processes responsible for the concerted evolution of the rDNA locus. Third, a study will be conducted of the differential replication of rDNA units in larval tissues. This latter experiment is of interest because Drosophila larvae grow normally even when large fractions of their rDNA units are inserted by under-replicating in polyploid tissues those units that have R1 and R2 insertions. Finally, to determine the tempo and developmental timing of retrotransposition, R1 and R2 activity will be monitored from one generation to the next. R1 retrotransposition is sufficiently high in the Harwich lines that new events can be monitored per generation. A search will be made for a lab strain that will allow similar studies with the R2 elements. These studies should provide an unprecedented understanding of the life cycle of two retrotransposable elements.

Because the target site for R1 and R2, the rDNA locus and the nucleolus that forms from it, plays a key role in all cellular metabolism, these studies will also provide insights into the evolution, replication and function of this critical cellular component.

DESCRIPTION (provided by applicant): Only a small fraction of eukaryotic genomes encode protein or is involved in the regulation of gene expression. A far larger fraction of these genomes has little or no function and is derived from the reverse transcription of RNA. For example, at least 40% of the human genome is composed of these reverse transcripts. The gradual accumulation of such insertions over time has played a significant role in shaping the size, structure and function of our genome. The family of retrotransposable elements known as LINEs generates the protein machinery responsible for most of these insertions. One of the best model systems in which to study LINEs is R2, an element that inserts in a sequence specific manner into a fraction of the hundreds of tandemly repeated 28S rRNA genes found in all higher organisms. The high degree of sequence specificity of the R2 integration reaction has enabled detailed biochemical studies of its mechanism. A critical but poorly understood aspect of this integration mechanism is how the R2 protein binds the two ends of the R2 RNA template used for reverse transcription. One series of experiments in this proposal is a detailed study of amino acid substitutions in the R2 protein that influence its ability to bind these RNA regions. These studies will contribute to a better understanding of how LINE elements are responsible for many of the insertions that occur in a more random manner throughout the human genome. Another goal of this proposal is to understand how R2 elements are regulated. The tandemly repeated rRNA genes (rDNA locus) form the nucleolus, the site of rRNA synthesis and ribosomal subunit assembly. While each R2 insertion disrupts the function of one 28S rRNA gene, an organism can survive as long as sufficient numbers of rRNA genes remain uninserted. Thus a second series of experiments is designed to determine how organisms are able to generate high levels of rRNA from uninserted genes while minimizing the expression of R2 elements from the otherwise identical inserted rRNA genes. Transcription of inserted rRNA genes gives rise to new R2 insertion. Using methods to monitor the transcription of specific R2 elements and to position these elements within the rDNA locus, the regions of the rDNA locus that are transcribed will be defined, and these transcribed regions monitored over time to determine how they are influenced by recombination and R2 element activity. Finally, mutations in a number of genes involved in chromosome structure and rRNA gene regulation will be tested in a third series of experiments to determine whether they affect the ability of the organism to differentiate between inserted and uninserted 28S genes. These experiments should reveal new insights into how transcription of the entire rDNA locus and its R2 elements are differentially regulated. PUBLIC HEALTH RELEVANCE: Transposable elements are extremely abundant in human genomes and are responsible directly or indirectly for many mutations associated with various genetic diseases and the onset of cancer. This research studies a model system that enables detailed studies of the insertion mechanism likely to be used by the most abundant, and only active, transposable element in humans. This research also studies the regulated expression of the many ribosomal RNA genes, genes that play a key role in all aspects of cellular metabolism.

All higher eukaryotic genomes are composed to a large measure of transposable elements that continually attempt to expand their numbers even further. These elements can be thought of as intracellular parasites. While it is generally assumed that transposable elements insert more or less at random, a growing number of elements have been shown to minimize their effects on the host by evolving site-specificity. The model systems used in this research are the R1 and R2 non-LTR retrotransposable elements, which insert specifically into the 28S rRNA genes of many animals but have been studied most extensively in insects. The hundreds of tandemly arranged rRNA genes (the rDNA loci) of eukaryotes undergo dynamic processes of recombination to eliminate variation within the locus. Yet throughout their history, R1 and R2 have stably maintained their presence in the rRNA genes indicating they are well-adapted to exploit the recombinational and regulatory mechanisms devised by the cell to synthesize rRNA. The objectives of this research are to study the rDNA genes and their R1and R2 insertions in several Drosophila species at three levels. The first objective is to define the changes that have occurred in specific rDNA loci over defined periods of time. Large segments of the rDNA loci from two replicate lines of D. melanogaster that have been separated by over 400 generations will be recovered on a series of overlapping recombinant DNA clones assembled using the 5' marked R1 and R2 elements as reference points. Detailed comparisons of the extended regions will enable a first view of the specific patterns of recombination and of the distribution of the retrotransposition events that have given rise to the many changes known to have occurred between these lines. Second, natural populations of D. simulans will be screened to determine the frequency with which R2 elements are active as well as to characterize the size and structure of the rDNA loci. These studies will be conducted in D. simulans because this sister species of D. melanogaster has no rDNA units on the Y chromosome and populations with active R2 elements are readily obtained. Third, public data made available through whole genome shot-gun sequencing efforts of 12 Drosophila species as well as those of other insects will be used to score the nucleotide variation within the rDNA loci of various animals. The goal will be to establish the approaches that can be used to compare the mechanism and efficiency of concerted evolution of the rDNA loci. These studies will enable a greater understanding of the fluctuation in size, the regulation of expression, and the recombinational processes that give rise to both the sequence uniformity and the segmental changes that occur in the rDNA locus over time. They will also increase our understanding of the delicate balance that is reached between higher organisms and these intracellular parasites.

The genomes of all higher organisms are in a constant battle with internal parasites called mobile or transposable elements. Between 10% and 90% of total genomic DNA from different organisms is composed of these elements. This research focuses on a model system that has the advantage that the mobile elements specifically insert into one location of the genome: the tandemly repeated ribosomal RNA genes (rDNA locus). The model organisms are several fruit fly species (Drosophila). The studies include short term laboratory experiments, population experiments, and the utilization of genomic sequence data to follow the mobile elements being inserted into and deleted from the rDNA locus, as well as how the rDNA locus changes as a result of these insertions. This knowledge will help researchers both control and exploit these elements for the advantage of mankind. The project will also serve to further the education of high school science teachers, undergraduates, and graduate students.