- Email: [email protected]
LCMV cDNA formation: which reverse transcriptase is responsible?
M E E T I N G R E P O RT S primary sequence) is then searched against databases of known threedimensional structures, and similarities are used to infer possible functions of the protein. The clinical fruits of the initial genomics effort are beginning to ripen. A leader in human cDNA sequencing, Human Genome Sciences, provided the most advanced clinical results. Human Genome Sciences focused its internal efforts on cDNA sequences of secreted proteins with therapeutic potential. Candidate secreted proteins were identified by homology searches of >50 000 cDNA sequences assembled from EST sequences. Each candidate secreted protein was produced and then screened for activity in disease models. Two of these candidates, KGF2 for wound healing and MPIF1 for chemoprotection, are now in clinical trials.
Integrated genomics companies, including Genome Therapeutics and Millennium Pharmaceuticals, focused more on the specifics of human disease projects involving a variety of techniques (e.g. differential display, expression profiling chips, antisense and high-throughput two-hybrid screens; Fig. 1). For example, Mark Osborne (Genome Therapeutics) described comparative EST sequencing of transcripts differentially regulated in a cell culture model of osteoblast differentiation. A number of known and novel differentially expressed transcripts were identified and the functions of these transcripts were studied with antisense technology. A log jam of information on sequences and expression profiles is accumulating at a variety of companies, with those having well-prepared bioinformatics systems expressing
relief. The quantity of data also requires careful statistical analysis because the detection of up to 40 000 transcripts with DNA-expression profiling can result in high variation of individual data points. In summary, the field is maturing as the first genomics-based drugs enter clinical trials and cDNA sequencing becomes saturated, and expanding with ever more detailed analysis of gene expression and genetic variation.
Tod M. Woolf [email protected] Director of Technology Development
Margaret Taylor [email protected] Manager of Collaborative Research Sequitur, Inc., Antisense Functional Genomics Group, 21 Birch Road, Suite B, Natick, MA 01760, USA.
LETTER
LCMV cDNA formation: which reverse transcriptase is responsible? In a recent paper Klenerman et al.1 demonstrated that infection of certain cell lines with a segmented RNA virus, lymphocytic choriomeningitis virus (LCMV), results in the formation of a low level of LCMV cDNA. This indicates that an endogenous source of reverse transcriptase (RT) in these cells is capable of copying the viral RNA into a DNA form (confirming a suggestion originally made by Zhdanov2 in 1975). Klenerman et al. speculate that either ‘an endogenous retrovirus, or a variety of other interspersed elements’ might provide the RT activity. Their data raise questions not only about the source of the RT, but also about how reverse transcription is primed and whether these cDNAs are inserted into the genome. Based on recent results from a variety of laboratories, we propose that the most likely source of RT in their study is the abundant family of mammalian non-LTR retrotransposons known as L1 elements.
The case for L1 RT comes from several lines of evidence. (1) There are an estimated 3000 full-length L1 elements in both the human and mouse genomes, many of which contain functional RTs and the capability to retrotranspose3,4. (2) Dhellin et al.5 used a PCR-based assay to show that overexpression of an L1 element in mouse or human cells results in cDNA formation for a variety of cellular transcripts. They found no evidence of cDNA synthesis after overexpression of either HIV or MuLV RT in these same cell lines, despite high levels of RT activity in cellular extracts. While previous studies have demonstrated ‘pseudogene formation’ in the presence of active L1s (Refs 6–8) or retroviruses9–11, the Dhellin et al. study provides a direct comparison of different RT sources and shows that efficient copying of cellular transcripts appears to be an L1-associated phenomenon. (3) We have previously shown that human L1 RT expressed in yeast can reverse TIG JUNE 1998 VOL. 14 NO. 6
Copyright © 1998 Elsevier Science Ltd. All rights reserved. 0168-9525/98/$19.00 PII: S0168-9525(98)01474-7
220
transcribe a marked antisense HIS3 RNA and that the resulting cDNA can be targeted to the site of a chromosomal double-strand break at the MAT locus8. By mapping the structure of numerous L1 RT-mediated cDNA insertions at the MAT locus, we have observed examples of complex events containing segments of multiple unlinked genes. Of particular relevance is an insertion containing ~100 bases of ‘W’ RNA (S-C. Teng and A. Gabriel, unpublished), an endogenous RNA virus of yeast that is found in both single-strand and double-strand linear RNA forms11,12. As with the Klenerman result, this is a clear example of cDNA formed from the genome of a non-retroviral RNA virus. Furthermore, its presence at the MAT locus shows that such patchwork cDNA products can indeed be integrated, at sites of chromosomal breaks. (4) In addition to cDNA insertion into chromosomal breaks, L1 elements and many other non-LTR retrotransposons appear to encode an endonuclease that can nick DNA, simultaneously providing a priming site for cDNA synthesis as well as a site for cDNA insertion6,12,13. This indicates another, more
LETTER orderly, pathway by which cDNA synthesis can be primed and lead to integration into host genomes via L1 elements. Taken together, these multiple lines of evidence indicate the ability of L1 RTs to promiscuously utilize a variety of RNA templates, including viral RNA sequences, and to insert the resulting cDNAs back into the genome by at least two different pathways. This relative indifference to the source of RNA templates distinguishes L1 RTs from the more fastidious retroviral RTs, and may help explain the observation that while all vertebrate genomes contain endogenous retroviral sequences, only mammalian genomes are home to both a plethora of processed pseudogenes and an abundance of L1 elements.
Abram Gabriel [email protected] Department of Molecular Biology and Biochemistry, Rutgers University, The Netherlands.
Shu-Chun Teng [email protected] Department of Molecular Biology, Princeton University, USA.
References 1 Klenerman, P., Hengartner, H. and Zinkernagel, R.M. (1997) Nature 390, 298–301 2 Zhdanov, V.M. (1975) Nature 256, 471–473 3 Sassaman, D.M. et al. (1997) Nat. Genet. 16, 37–43 4 Naas, T.P. et al. (1998) EMBO J. 17, 590–597
5 Dhellin, O., Maestre, J. and Heidmann, T. (1997) EMBO J. 16, 6590–6602 6 Moran, J.V. et al. (1996) Cell 87, 917–927 7 Dombroski, B.A. et al. (1994) Mol. Cell. Biol. 14, 4485–4492 8 Teng, S-C., Kim, B. and Gabriel, A. (1996) Nature 383, 641–644 9 Dornburg, R. and Temin, H.M. (1990) Mol. Cell. Biol. 10, 68–74 10 Levine, K.L. et al. (1990) Mol. Cell. Biol. 10, 1891–1900 11 Carlton, M.B.L., Colledge, W.H. and Evans, M.J. (1995) Mamm. Genome 6, 90–95 12 Feng, Q.H., Moran, J.V., Kazazian, H.H.J. and Boeke, J.D. (1996) Cell 87, 905–916 13 Luan, D.D., Korman, M.H., Jakubczak, J.L. and Eickbush, T.H. (1993) Cell 72, 595–605
MONITOR
A ‘digest’ of some recent papers of interest in the primary journals
Argonaute catapults new gene family to prominence AGO1 defines a novel locus of Arabidopsis controlling leaf development Bohmert, K. et al. EMBO J. 17, 170–180 Role of the ZWILLE gene in the regulation of central shoot meristem cell fate during Arabidopsis embryogenesis Moussian, B. et al. EMBO J. 7, 1799–1809 Organogenesis in multicellular organisms involves the coordinated development of individual cells to produce tissues and organs. Natural selection often results in a single system becoming co-opted to coordinate different events during development or similar processes in different organisms. This results in gene families that regulate diverse developmental processes. Two recent papers describe genes affecting different aspects of development in Arabidopsis but have a high degree of similarity (75% identity over most of the protein). Both of these genes contain large regions of similarity to genes in rice, humans, rats and a family of nine genes in Caenorhabditis elegans. No related sequences occur in yeast or bacteria. This suggests a new
gene family of regulators of multicellular development that are involved in the regulation of several different pathways in each organism. Sequence analysis suggests that the proteins encoded are soluble in the cytoplasm but no known motifs have been identified, so the only clues to the function of the genes come from the mutant phenotypes of the Arabidopsis genes. These phenotypes support a role for this gene family in coordinating and specifying groups of cells. zwille mutant plants lack a shoot meristem although ectopic shoot meristems can form and produce normal shoots. In situ hybridization shows that the ZWILLE (ZLL) gene product is required to maintain expression of the SHOOT MERISTEMLESS gene (required for TIG JUNE 1998 VOL. 14 NO. 6
221
meristem function) during late embryogenesis. argonaute plants are defective in organ development. Leaves, sepals and petals organs are not laterally flattened, stamens lack anthers and dual carpels are common. A role for ARGONAUTE (AGO) in the regulation of meristematic cells is suggested by the occurrence of ectopic meristems in plants overexpressing AGO. Northerns show that AGO is expressed at low levels in all tissues of the plant. In situ hybridizations of AGO mRNA and the generation of double mutants will reveal whether the genes are coexpressed and functionally redundant. The proteins are most similar to each other and to other members of the gene family at the C-terminus. The unconserved glycine-rich N-terminus of AGO and the proline-rich N-terminus of ZLL are presumably involved in AGO- and ZLL-specific functions. The conserved regions are also required for proteins to be functional in specific pathways, and might represent common pathways acting in different organisms. Hopefully, animal biologists will not be too long catching up with the plants and providing mutant phenotypes of other members of this gene family. ✍
Integrated genomics companies, including Genome Therapeutics and Millennium Pharmaceuticals, focused more on the specifics of human disease projects involving a variety of techniques (e.g. differential display, expression profiling chips, antisense and high-throughput two-hybrid screens; Fig. 1). For example, Mark Osborne (Genome Therapeutics) described comparative EST sequencing of transcripts differentially regulated in a cell culture model of osteoblast differentiation. A number of known and novel differentially expressed transcripts were identified and the functions of these transcripts were studied with antisense technology. A log jam of information on sequences and expression profiles is accumulating at a variety of companies, with those having well-prepared bioinformatics systems expressing
relief. The quantity of data also requires careful statistical analysis because the detection of up to 40 000 transcripts with DNA-expression profiling can result in high variation of individual data points. In summary, the field is maturing as the first genomics-based drugs enter clinical trials and cDNA sequencing becomes saturated, and expanding with ever more detailed analysis of gene expression and genetic variation.
Tod M. Woolf [email protected] Director of Technology Development
Margaret Taylor [email protected] Manager of Collaborative Research Sequitur, Inc., Antisense Functional Genomics Group, 21 Birch Road, Suite B, Natick, MA 01760, USA.
LETTER
LCMV cDNA formation: which reverse transcriptase is responsible? In a recent paper Klenerman et al.1 demonstrated that infection of certain cell lines with a segmented RNA virus, lymphocytic choriomeningitis virus (LCMV), results in the formation of a low level of LCMV cDNA. This indicates that an endogenous source of reverse transcriptase (RT) in these cells is capable of copying the viral RNA into a DNA form (confirming a suggestion originally made by Zhdanov2 in 1975). Klenerman et al. speculate that either ‘an endogenous retrovirus, or a variety of other interspersed elements’ might provide the RT activity. Their data raise questions not only about the source of the RT, but also about how reverse transcription is primed and whether these cDNAs are inserted into the genome. Based on recent results from a variety of laboratories, we propose that the most likely source of RT in their study is the abundant family of mammalian non-LTR retrotransposons known as L1 elements.
The case for L1 RT comes from several lines of evidence. (1) There are an estimated 3000 full-length L1 elements in both the human and mouse genomes, many of which contain functional RTs and the capability to retrotranspose3,4. (2) Dhellin et al.5 used a PCR-based assay to show that overexpression of an L1 element in mouse or human cells results in cDNA formation for a variety of cellular transcripts. They found no evidence of cDNA synthesis after overexpression of either HIV or MuLV RT in these same cell lines, despite high levels of RT activity in cellular extracts. While previous studies have demonstrated ‘pseudogene formation’ in the presence of active L1s (Refs 6–8) or retroviruses9–11, the Dhellin et al. study provides a direct comparison of different RT sources and shows that efficient copying of cellular transcripts appears to be an L1-associated phenomenon. (3) We have previously shown that human L1 RT expressed in yeast can reverse TIG JUNE 1998 VOL. 14 NO. 6
Copyright © 1998 Elsevier Science Ltd. All rights reserved. 0168-9525/98/$19.00 PII: S0168-9525(98)01474-7
220
transcribe a marked antisense HIS3 RNA and that the resulting cDNA can be targeted to the site of a chromosomal double-strand break at the MAT locus8. By mapping the structure of numerous L1 RT-mediated cDNA insertions at the MAT locus, we have observed examples of complex events containing segments of multiple unlinked genes. Of particular relevance is an insertion containing ~100 bases of ‘W’ RNA (S-C. Teng and A. Gabriel, unpublished), an endogenous RNA virus of yeast that is found in both single-strand and double-strand linear RNA forms11,12. As with the Klenerman result, this is a clear example of cDNA formed from the genome of a non-retroviral RNA virus. Furthermore, its presence at the MAT locus shows that such patchwork cDNA products can indeed be integrated, at sites of chromosomal breaks. (4) In addition to cDNA insertion into chromosomal breaks, L1 elements and many other non-LTR retrotransposons appear to encode an endonuclease that can nick DNA, simultaneously providing a priming site for cDNA synthesis as well as a site for cDNA insertion6,12,13. This indicates another, more
LETTER orderly, pathway by which cDNA synthesis can be primed and lead to integration into host genomes via L1 elements. Taken together, these multiple lines of evidence indicate the ability of L1 RTs to promiscuously utilize a variety of RNA templates, including viral RNA sequences, and to insert the resulting cDNAs back into the genome by at least two different pathways. This relative indifference to the source of RNA templates distinguishes L1 RTs from the more fastidious retroviral RTs, and may help explain the observation that while all vertebrate genomes contain endogenous retroviral sequences, only mammalian genomes are home to both a plethora of processed pseudogenes and an abundance of L1 elements.
Abram Gabriel [email protected] Department of Molecular Biology and Biochemistry, Rutgers University, The Netherlands.
Shu-Chun Teng [email protected] Department of Molecular Biology, Princeton University, USA.
References 1 Klenerman, P., Hengartner, H. and Zinkernagel, R.M. (1997) Nature 390, 298–301 2 Zhdanov, V.M. (1975) Nature 256, 471–473 3 Sassaman, D.M. et al. (1997) Nat. Genet. 16, 37–43 4 Naas, T.P. et al. (1998) EMBO J. 17, 590–597
5 Dhellin, O., Maestre, J. and Heidmann, T. (1997) EMBO J. 16, 6590–6602 6 Moran, J.V. et al. (1996) Cell 87, 917–927 7 Dombroski, B.A. et al. (1994) Mol. Cell. Biol. 14, 4485–4492 8 Teng, S-C., Kim, B. and Gabriel, A. (1996) Nature 383, 641–644 9 Dornburg, R. and Temin, H.M. (1990) Mol. Cell. Biol. 10, 68–74 10 Levine, K.L. et al. (1990) Mol. Cell. Biol. 10, 1891–1900 11 Carlton, M.B.L., Colledge, W.H. and Evans, M.J. (1995) Mamm. Genome 6, 90–95 12 Feng, Q.H., Moran, J.V., Kazazian, H.H.J. and Boeke, J.D. (1996) Cell 87, 905–916 13 Luan, D.D., Korman, M.H., Jakubczak, J.L. and Eickbush, T.H. (1993) Cell 72, 595–605
MONITOR
A ‘digest’ of some recent papers of interest in the primary journals
Argonaute catapults new gene family to prominence AGO1 defines a novel locus of Arabidopsis controlling leaf development Bohmert, K. et al. EMBO J. 17, 170–180 Role of the ZWILLE gene in the regulation of central shoot meristem cell fate during Arabidopsis embryogenesis Moussian, B. et al. EMBO J. 7, 1799–1809 Organogenesis in multicellular organisms involves the coordinated development of individual cells to produce tissues and organs. Natural selection often results in a single system becoming co-opted to coordinate different events during development or similar processes in different organisms. This results in gene families that regulate diverse developmental processes. Two recent papers describe genes affecting different aspects of development in Arabidopsis but have a high degree of similarity (75% identity over most of the protein). Both of these genes contain large regions of similarity to genes in rice, humans, rats and a family of nine genes in Caenorhabditis elegans. No related sequences occur in yeast or bacteria. This suggests a new
gene family of regulators of multicellular development that are involved in the regulation of several different pathways in each organism. Sequence analysis suggests that the proteins encoded are soluble in the cytoplasm but no known motifs have been identified, so the only clues to the function of the genes come from the mutant phenotypes of the Arabidopsis genes. These phenotypes support a role for this gene family in coordinating and specifying groups of cells. zwille mutant plants lack a shoot meristem although ectopic shoot meristems can form and produce normal shoots. In situ hybridization shows that the ZWILLE (ZLL) gene product is required to maintain expression of the SHOOT MERISTEMLESS gene (required for TIG JUNE 1998 VOL. 14 NO. 6
221
meristem function) during late embryogenesis. argonaute plants are defective in organ development. Leaves, sepals and petals organs are not laterally flattened, stamens lack anthers and dual carpels are common. A role for ARGONAUTE (AGO) in the regulation of meristematic cells is suggested by the occurrence of ectopic meristems in plants overexpressing AGO. Northerns show that AGO is expressed at low levels in all tissues of the plant. In situ hybridizations of AGO mRNA and the generation of double mutants will reveal whether the genes are coexpressed and functionally redundant. The proteins are most similar to each other and to other members of the gene family at the C-terminus. The unconserved glycine-rich N-terminus of AGO and the proline-rich N-terminus of ZLL are presumably involved in AGO- and ZLL-specific functions. The conserved regions are also required for proteins to be functional in specific pathways, and might represent common pathways acting in different organisms. Hopefully, animal biologists will not be too long catching up with the plants and providing mutant phenotypes of other members of this gene family. ✍