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Allan Award Lecture: On Jumping Fields and “Jumping Genes”

2009; Elsevier BV; Volume: 84; Issue: 2 Linguagem: Inglês

10.1016/j.ajhg.2009.01.003

1537-6605

Haig H. Kazazian,

CRISPR and Genetic Engineering

First, let me thank the American Society of Human Genetics for this great honor. For the 40 years that I have attended the ASHG meeting, I have been impressed with the significance of the Allan Award in honoring research accomplishment in human genetics. This is a very important day for me, and I greatly appreciate your recognition. Before going further, I'd like to say that there is someone we all miss very much at this meeting, Victor McKusick. He did so much for the field of human genetics and for our society. Victor meant a lot to me during my 25 years at Johns Hopkins, both personally and professionally. His intellect was extraordinary, but he never flaunted it. What a wonderful man he was! Now, I'd like to tell you about my journey in human genetics, hitting the high points and providing a few anecdotes and philosophical comments. I'll also stress the role of teamwork and interaction with colleagues in the research process. I'll end by discussing what has gotten me excited in the lab presently. In doing research, I've tried to work on important problems, yet ones that were solvable using available methods. I've also tried not to follow the crowd, but in the words of Robert Frost to take “the road less traveled by, And that has made all the difference.” Every researcher is a gambler, but the smart gambler likes odds that are not too long. In research, problems with odds of success about 5 or 10:1 are good problems. If the odds are 2:1, the problem is probably not so important. If the odds are 100:1, the problem is too risky. I'm going to tell you about my research course starting from globin regulation in the thalassemias, then turning to “jumping genes,” and how even though those subjects seem quite disparate, the course followed a logical route. Because of time constraints, I won't speak about other projects, namely, cystic fibrosis work by Garry Cutting, schizophrenia research with Ann Pulver, characterization of hemophilia A mutations with Stelios Antonarakis, and gene therapy of hemophilia A in the mouse and dog models by Rita Sarkar and Denise Sabatino. My first research experience was at Dartmouth Medical School in the summers of 1958 to 1960. Before starting medical school after my junior year in 1958, I asked the Dean whether I should work as a hospital orderly. He told me to spend the summer doing biochemical research with Lafayette Noda on rabbit creatine kinase. The following two summers I worked with Lucille Smith on bacterial electron transport. However, after transferring to Hopkins for the last 2 years of medical school, I remained unconvinced that research was my career path. I enjoyed an elective in child psychiatry, and decided to train in pediatrics. As a fourth year medical student in 1962, I took an elective taught by Barton Childs in genetics. Parenthetically, this was the only time that 8 week course was given. Each of six students and two faculty gave a seminar on a literature topic. I remember telling Barton in his office that I knew nothing about genetics, and he said, “Don't worry, you'll learn.” My seminar was on the cytogenetic abnormalities that were being published almost weekly at that time, and I got hooked on human genetics. At that time, I also got hooked on Lilli, my loving wife of many years. After an internship in pediatrics at the University of Minnesota, I decided to do a genetics fellowship after only one year of residency. I wrote Barton asking him to suggest places to train. He mentioned a few places including Hopkins, where he had recently obtained an NIH training grant. After reading the ground-breaking Davidson, Nitowsky, and Childs paper1Davidson R.G. Nitowski H.M. Childs B. Demonstration of two populations of cells in the human female heterozygous for glucose=6 phosphate dehydrogenase variants.Proc. Natl. Acad. Sci. USA. 1963; 50: 481-485Crossref PubMed Scopus (140) Google Scholar in PNAS demonstrating the validity of the Lyon hypothesis in humans, I decided to train with Dr. Childs. At Hopkins, Childs suggested that I study genetic variation in fruit flies in Bill Young's lab, and we were successful in demonstrating that X chromosome dosage compensation in flies does not proceed by the same X inactivation mechanism used by mammals.2Kazazian Jr., H.H. Young W.J. Childs B. X-linked 6-phosphogluconate dehydrogenase in Drosophila: Subunit associations.Science. 1965; 150: 1601-1602Crossref PubMed Scopus (40) Google Scholar That was the gist of my first paper in 1965. After 20 months at Hopkins and with the U.S. Army breathing down my neck, I joined the U.S. Public Health Service at the NIH. Luckily, I found a position with Harvey Itano largely due to a recommendation from my Dartmouth mentor, Lafayette Noda. Note the Japanese-American connection. With Itano, I did research on the regulation of globin synthesis in humans. Then Bob Cooke, Chair of Pediatrics at Hopkins, offered me a faculty position, but I asked Dr. Cooke to let me finish a third year of pediatric training at Hopkins first. During elective time in that residency year, I wrote my first successful RO1 application on globin gene regulation. So I had funding when I joined the faculty in 1969. I started working on separating the alpha and beta globin messenger RNAs in a small operation, a technician and me. However, I occupied the lab designated for Childs, so the space was ample. I knew that in order to succeed competing against larger, smarter labs I would need to spend most of my time on research. My goal was to emulate the high-quality research in the basic sciences at Hopkins. Shortly thereafter, Mike Kaback started his Tay-Sachs screening program in the Baltimore Jewish community.3Kaback M. Lim-Steele J. Dabholkar D. Brown D. Levy N. Zeiger K. Tay-Sachs disease–carrier screening, prenatal diagnosis, and the molecular era. An international perspective, 1970 to 1993. The International TSD Data Collection Network.JAMA. 1993; 270: 2307-2315Crossref PubMed Scopus (202) Google Scholar He suggested that if adult hemoglobin were produced in the fetus, one might similarly be able to do carrier screening and prenatal diagnosis for sickle cell anemia. I was keen to look into this, and Morley Hollenberg, an MD student with an Oxford DPhil, quickly found hemoglobin A synthesis in the midtrimester fetus using techniques that I had learned at NIH with Itano.4Hollenberg M.D. Kaback M.M. Kazazian Jr., H.H. Adult hemoglobin synthesis by reticulocytes from the human fetus at midtrimester.Science. 1971; 174: 698-702Crossref PubMed Scopus (57) Google Scholar Prenatal detection of hemoglobin A was my entree into the prenatal diagnosis field. In the early 1970s I continued to work on the mRNA imbalance in the alpha- and beta-thalassemias. In 1975, John Phillips, a former chief resident at Boston Children's, joined the lab. John did lovely studies of normal globin mRNA ratios,5Phillips III, J.A. Snyder P.G. Kazazian Jr., H.H. Ratios of α to β globin mRNA and regulation of globin synthesis in reticulocytes.Nature. 1977; 269: 442-445Crossref PubMed Scopus (12) Google Scholar but his breakthrough came through a fortuitous chain of events. In early 1978, Ned Boyer, a senior faculty in McKusick's Division of Medical Genetics, sent Alan Scott, a postdoc, to Alec Jeffreys's lab in England to learn Southern blotting. After Alan returned, Phillips suggested and I agreed that he learn Southern blotting from Alan. Y.W. Kan had recently found a HpaI polymorphic site 3′ of the beta-globin gene, and demonstrated its usefulness in prenatal diagnosis of sickle cell anemia.6Kan Y.W. Dozy A.M. Antenatal diagnosis of sickle-cell anaemia by D.N.A. analysis of amniotic-fluid cells.Lancet. 1978; 2: 910-912Abstract PubMed Scopus (259) Google Scholar Soon thereafter, Jeffreys found two polymorphic HindIII sites in the gamma-globin genes.7Jeffreys A.J. DNA sequence variants in the G gamma-, A gamma-, delta- and beta-globin genes of man.Cell. 1979; 18: 1-10Abstract Full Text PDF PubMed Scopus (295) Google Scholar Phillips used Southern blotting to find extended linkage disequilibrium involving the Beta S mutation and the HpaI and HindIII sites.8Phillips III, J.A. Panny S.R. Kazazian Jr., H.H. Boehm C.D. Scott A.F. Smith K.D. Prenatal diagnosis of sickle cell anemia by restriction endonuclease analysis: Hind III polymorphisms in γ-globin genes extend test applicability.Proc. Natl. Acad. Sci. USA. 1980; 77: 2853-2856Crossref PubMed Scopus (54) Google Scholar Out of eight possibilities for the three sites, there were only four varieties of Beta S-bearing chromosomes. The 60% with the HpaI site lacked both HindIII sites, whereas the 40% without the HpaI site usually contained one HindIII site. This setup allowed an increase in precise prenatal detection of sickle cell anemia by linkage analysis from 60% to 85%.8Phillips III, J.A. Panny S.R. Kazazian Jr., H.H. Boehm C.D. Scott A.F. Smith K.D. Prenatal diagnosis of sickle cell anemia by restriction endonuclease analysis: Hind III polymorphisms in γ-globin genes extend test applicability.Proc. Natl. Acad. Sci. USA. 1980; 77: 2853-2856Crossref PubMed Scopus (54) Google Scholar This paper was crucial to the thinking that went into our later work characterizing mutations in beta-thalassemia. In 1979, we hired Corinne Boehm to run our molecular diagnosis program, which consisted only of prenatal diagnosis of sickle cell anemia by linkage analysis. In July 1980, Antonarakis entered the picture. He had written me asking for a genetics training position. I had no extra money to pay him, but he said he'd come without pay. His letter languished on my desk until another colleague, George Stamatoyannopoulos, called from Seattle to push for Antonarakis. George was very persuasive, so I hired Stelios without pay. Once he arrived, he got $5000 from the Greek church in Baltimore. On Christmas day 1980, at a family get-together in Detroit, I received a call from the Armenian industrialist and family friend I had solicited. He said he'd provide another $5000 for Antonarakis. So Stelios was paid something. Antonarakis used Southern blotting to find more RFLPs in the beta globin cluster. He found three new HincII site polymorphisms in the epsilon and pseudo-beta regions. Meanwhile, Kan had found a polymorphic BamHI site 3′ of the beta gene.9Kan Y.W. Lee K.Y. Furbetta M. Angius A. Cao A. Polymorphism of DNA sequence in the beta-globin gene region. Application to prenatal diagnosis of beta 0 thalassemia in Sardinia.N. Engl. J. Med. 1980; 302: 185-188Crossref PubMed Scopus (116) Google Scholar Then Stelios found a polymorphic AvaII site in the beta gene itself. Parenthetically, these RFLPs became SNPs 20 years later. In studying these polymorphic sites in families, there emerged an interesting pattern. There was clear linkage disequilibrium of the five sites 5′ to the delta gene (only three common polymorphic patterns were present out of a potential 32). There was also linkage disequilibrium for the AvaII and BamHI sites in and 3′ of the beta gene (three patterns out of a predicted four).10Antonarakis S.E. Boehm C.D. Giardina P.J.V. Kazazian Jr., H.H. Non-random association of polymorphic restriction sites in the β-globin gene cluster.Proc. Natl. Acad. Sci. USA. 1982; 79: 137-141Crossref PubMed Scopus (249) Google Scholar However, the two regions of disequilibrium combined together randomly, i.e., the three 5′ patterns randomly associated with the three 3′ patterns. This suggested that within the intervening ∼12 kb was a recombination hot spot.10Antonarakis S.E. Boehm C.D. Giardina P.J.V. Kazazian Jr., H.H. Non-random association of polymorphic restriction sites in the β-globin gene cluster.Proc. Natl. Acad. Sci. USA. 1982; 79: 137-141Crossref PubMed Scopus (249) Google Scholar Indeed, a number of recombination events have been seen in this hot-spot region (Figure 1). One day in 1981, I asked the lab what to call the pattern of polymorphic restriction sites on a chromosome. Corinne Boehm suggested the term “haplotype” after the usage for HLA haplotypes coined a few years earlier by Ceppelini. “Haplotype” in the DNA context appeared for the first time in January 1982.10Antonarakis S.E. Boehm C.D. Giardina P.J.V. Kazazian Jr., H.H. Non-random association of polymorphic restriction sites in the β-globin gene cluster.Proc. Natl. Acad. Sci. USA. 1982; 79: 137-141Crossref PubMed Scopus (249) Google Scholar Later, we found a polymorphic TaqI site upstream of the epsilon gene that was not in linkage disequilibrium with the sites in the beta globin cluster, and thus the region extending from the epsilon gene to the delta gene constituted the first “haplotype block” as analyzed by Chakravarti.11Chakravarti A. Buetow K.H. Antonarakis S.E. Waber P.G. Boehm C.D. Kazazian Jr., H.H. Non-uniform recombination within the human β-globin gene cluster.Am. J. Hum. Genet. 1984; 36: 1239-1284PubMed Google Scholar This was my introduction to genomic analysis to which I later returned in studies of mobile DNA. In 1980, we used haplotypes to carry out prenatal diagnosis of beta-thalassemia in families with a previously affected child.12Kazazian Jr., H.H. Phillips III, J.A. Boehm C.D. Vik T. Mahoney M.J. Ritchey A.K. Prenatal diagnosis of β-thalassemia by amniocentesis: Linkage analysis of multiple polymorphic restriction endonuclease sites.Blood. 1980; 56: 926-930PubMed Google Scholar However, we wondered whether the lesson of restriction-site polymorphisms and their linkage disequilibrium with the Beta S mutation would carry over to beta-thalassemia. By this time, three labs had sequenced six beta-thalassemia genes, but only two different mutations had been found, each one three times. Since we had few beta-thalassemia patients at Hopkins, I called Pat Giardina, Director of the Thalassemia Clinic at New York Hospital. She had a large patient population, and could get blood samples on families. To do haplotype analysis, we needed blood on trios, the patient and both parents. Amazingly, within 1 month Giardina had sent blood on some 30 trios which the lab quickly haplotyped. Around this time I contacted Stuart Orkin, a friend and globin colleague, at Boston Children's. Stu was interested in sequencing beta-thalassemia genes. I thought that perhaps different normal chromosome backgrounds were hit by different mutations (Figure 2), and because the beta-globin cluster was so small there had been insufficient time in generations for the mutations to move to different haplotypes by recombination. Thus, we might have a neat analytical way to characterize all the common mutations causing beta-thalassemia. I suggested to Stu that I send him specific beta-thalassemia DNAs each containing beta-thalassemia mutations on different haplotypes. After sending the first three DNAs to Boston, I remember Stu's call a few weeks later. He had sequenced three beta genes of interest, and they all had different mutations that had not been seen previously. I nearly fainted from the excitement! We quickly finished cloning and sequencing the beta-thalassemia gene from nine different haplotypes in Mediterranean patients, finding eight different mutations.13Orkin S.H. Kazazian Jr., H.H. Antonarakis S.E. Goff S.C. Boehm C.D. Sexton J.P. Waber P.G. Giardina P.J.V. Linkage of β-thalassemia mutations and β-globin gene polymorphisms with DNA polymorphisms in the human β-globin gene cluster.Nature. 1982; 296: 627-631Crossref PubMed Scopus (680) Google Scholar At this time, Orkin and I were communicating new information daily by phone. Stu came to Baltimore and stayed at our home as we finished the paper. Later, we used haplotyping followed by cloning and sequencing at Hopkins to characterize the common beta-thalassemia alleles in Asian Indians, Chinese, and African-American patients.14Kazazian Jr., H.H. Orkin S.H. Antonarakis S.E. Sexton J.P. Boehm C.D. Goff S.C. Waber P.G. Molecular characterization of seven β-thalassemia mutations in Asian Indians.EMBO J. 1984; 3: 593-596Crossref PubMed Scopus (155) Google Scholar, 15Cheng T.C. Orkin S.H. Antonarakis S.E. Potter M.J. Sexton J.P. Giardina P.J.V. Li A. Kazazian Jr., H.H. β-thalassemia in Chinese: Use of in vivo RNA analysis and oligonucleotide hybridization in systematic characterization of molecular defects.Proc. Natl. Acad. Sci. USA. 1984; 81: 2821-2825Crossref PubMed Scopus (138) Google Scholar, 16Antonarakis S.E. Orkin S.H. Cheng T.-c. Scott A.F. Sexton J.P. Trusko S. Charache S. Kazazian Jr., H.H. β-thalassemia in American Blacks: Novel mutations in the TATA box and IVS-2 acceptor splice site.Proc. Natl. Acad. Sci. USA. 1984; 81: 1154-1158Crossref PubMed Scopus (132) Google Scholar The procedure of sequencing beta-thalassemia genes from different haplotypes worked well. A new allele was discovered 80% of the time. We characterized roughly 40 alleles that acted at a variety of steps in gene action: transcription, RNA splicing, RNA processing, translation, and protein stability (Figure 3). In 1984, Orkin and I reviewed the topic.17Orkin S.H. Kazazian Jr., H.H. Mutation and polymorphism of the human β-globin gene and its surrounding DNA.Annu. Rev. Genet. 1984; 18: 131-171Crossref PubMed Scopus (237) Google Scholar Beta-thalassemia was the first disorder caused by multiple mutations that was essentially completely characterized at the molecular level.Figure 3β-Thalassemia Mutations in Different Steps in Gene ActionShow full captionβ-thalassemia mutations in different steps in gene action, including transcription, RNA processing (Cap and polyadenylation), RNA splicing, translation (initiation codon, nonsense codons, and frameshifts), and protein stability.View Large Image Figure ViewerDownload Hi-res image Download (PPT) β-thalassemia mutations in different steps in gene action, including transcription, RNA processing (Cap and polyadenylation), RNA splicing, translation (initiation codon, nonsense codons, and frameshifts), and protein stability. However, it looked like the beta-thalassemia project would soon become humdrum as thalassemia alleles were characterized in different ethnic groups. Orkin and I discussed this problem at the 1983 ASHG meeting in Newport Beach, Virginia. He had found his future niche in transcription factors in hematopoiesis beginning with GATA-1. I was still looking for mine, and it would come, but not until the summer of 1987. In 1985, there was another lucky occurrence. Henry Erlich and Norm Arnheim wanted me to consult for Cetus in the East Bay, but I declined because the travel distance was too great and I foolishly thought I wouldn't learn anything new at the company. Boy, was I wrong! In 1985, our daughter, Sonya, decided to attend Stanford, and it became convenient to consult at Cetus when visiting Palo Alto. In September 1985, Lilli and I took Sonya to Stanford, and while at Cetus Henry and Norm ushered me into a small conference room to hear a wild idea from Kerry Mullis. This was my introduction to PCR, but although Randy Saiki had made some progress, the idea hadn't proceeded very far. Over the following year, we had little success in the lab with PCR. But Lilli talked me into returning to Stanford for sophomore parent's weekend in October, 1986. Now upon entering Erlich's office, Henry's excitement was palpable. David Gelfand had cloned Taq polymerase and the resultant PCR bands were incredible. We then used PCR with Taq polymerase to sequence beta-thalassemia genes directly from leukocyte DNA to discover mutations.18Wong C. Dowling C.E. Saiki R.K. Higuchi R.G. Erlich H.A. Kazazian Jr., H.H. Characterization of β-thalassemia mutations using direct genomic sequencing of amplified single copy DNA.Nature. 1987; 330: 384-386Crossref PubMed Scopus (360) Google Scholar From 1982 to 1987 haplotype analysis had aided mutation discovery in beta globin, alpha globin, phenyalanine hydroxylase, and LDL receptor genes. Now it was passé! PCR could be used to sequence mutations directly from leukocyte DNA in any gene whose sequence was known. Meanwhile, in 1984, to explore the full spectrum of mutations causing disease, we wanted to work on a large gene, whose mutations unlike those in beta-globin were not under selection, and where nearly every unrelated individual probably had a different mutation. The ideal gene appeared when Gitscher and colleagues at Genentech and Wozney, Toole, and colleagues at GI cloned the factor VIII gene that is mutated in hemophilia A.19Gitschier J. Wood W.I. Goralka T.M. Wion K.L. Chen E.Y. Eaton D.H. Vehar G.A. Capon D.J. Lawn R.M. Characterization of the human factor VIII gene.Nature. 1984; 312: 326-330Crossref PubMed Scopus (693) Google Scholar, 20Toole J.J. Knopf J.L. Wozney J.M. Sultzman L.A. Buecker J.L. Pittman D.D. Kaufman R.J. Brown E. Shoemaker C. Orr E.C. et al.Molecular cloning of a cDNA encoding human antihaemophilic factor.Nature. 1984; 312: 342-347Crossref PubMed Scopus (639) Google Scholar Here was the perfect gene, very large (nearly 200 kb) and X-linked, and Haldane had predicted in 1938 that every unrelated affected male would have a different mutation (Of course, the recurrent FVIII inversion found later by Gitscher made that prediction incorrect.21Lakich D. Kazazian Jr., H.H. Antonarakis S.E. Gitschier J. Inversions disrupting the factor VIII gene as a common cause of severe hemophilia A.Nat. Genet. 1993; 5: 236-241Crossref PubMed Scopus (655) Google Scholar). In 1984, Antonarakis and I traveled to GI in Boston, and obtained the FVIII cDNA for mutation analysis. In May 1987, Hagop Youssoufian, a genetics fellow, made a key discovery. He had been characterizing FVIII mutations in 240 hemophilia A patients by Southern blotting using FVIII cDNA probes. Two patients had abnormal restriction fragments, suggesting that their mutations were insertions. Although Hagop was leaving in July, he persuaded me to let him clone a portion of one insertion. One Monday morning, Hagop told me that over the weekend he had succeeded, blotted the clone with L1 and Alu probes, and found the insertion was part of an L1 repetitive element. Immediately, I thought this is the problem that I've been looking for. L1 is an insertional mutagen that can jump into genes, disrupt them, and cause disease. “I'm going to work on human transposable elements.” At the time, I knew something about L1s. At Hopkins and Cold Spring Harbor, I had heard Maxine Singer speak on repeat sequences in human DNA. Moreover, Allan Scott had joined the faculty in Pediatric Genetics, and after cloning and sequencing a number of human L1s, he had built an important consensus sequence for the full-length 6 kb L1.22Scott A.F. Schmeckpeper B.J. Abdelrazik M. Comey C.T. O'Hara B. Rossiter J.P. Cooley T. Heath P. Smith K.D. Margolet L. Origin of the human L1 elements: proposed progenitor genes deduced from a consensus DNA sequence.Genomics. 1987; 1: 113-125Crossref PubMed Scopus (247) Google Scholar Scott's consensus sequence had two open reading frames, called ORF1 and ORF2. ORF2 contained a sequence that when translated had similarity to the reverse transcriptases of retroviruses. It was thought that these sequences were retrotransposons, elements that could duplicate themselves through reverse transcription of an RNA intermediate. After Youssoufian left, Corinne Wong took up the project of characterizing the two L1 insertions, both of which were 5′ truncated. Meanwhile, at Singer's suggestion we found that the inserted L1s came from a special subfamily of transcribed L1 elements. Shown in Figure 4 are the parents and the young man (patient JH-27) with hemophilia A who had an L1 insertion. In Figure 5 is the 3.8 kb insertion of a 5′ truncated L1 into exon 14 of JH-27's factor VIII gene. Note the insertion was not present in his mother, meaning that it occurred either in her germ cells or early in his development. We published this paper in early 198823Kazazian Jr., H.H. Wong C. Youssoufian H. Scott A.F. Phillips D. Antonarakis S.E. A novel mechanism of mutation in man: Hemophilia A due to de novo insertion of L1 sequences.Nature. 1988; 332: 164-166Crossref PubMed Scopus (597) Google Scholar and I entered the “black hole” of transposable elements.Figure 5L1 Insertion into Exon 14 of the Factor VIII Gene in Patient JH-27Show full captionThe 3.8 kb 5′ truncated insertion ends in a poly A tract and is bracketed by short target-site duplications. In the pedigree, the insertion is not seen in the mother so it occurred either in one of her germ cells or in early embryogenesis of JH-27.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The 3.8 kb 5′ truncated insertion ends in a poly A tract and is bracketed by short target-site duplications. In the pedigree, the insertion is not seen in the mother so it occurred either in one of her germ cells or in early embryogenesis of JH-27. In early 1988, Beth Dombroski joined the lab as a postdoc, and we decided to try to isolate the full-length 6 kb L1 precursor of the JH-27 insertion. Our premise was that the precursor would have the identical sequence as the insertion over the 3.8 kb of the insertion, and would be an active mobile element. Of course, if I had known more about mobile elements, I would have realized that this premise depended on the element acting in cis, i.e., mobilizing a copy of itself into a new genomic site, not in trans, i.e., mobilizing a copy of another element as is the case for nearly every other transposable element. Thus, with more experience in the field I may not have done the experiment. I compared Alan Scott's L1 consensus sequence with the sequence of the 3.8 kb JH-27 insertion, and found a region of 20 nucleotides (nts) at roughly nt. 4200 in the 6 kb L1 consensus sequence that differed from the insertion by 3 nts. We made an oligonucleotide (JH-27) containing the 20 nts from the insertion sequence, and Beth carried out the blot against DNA digested with BamHI from JH-27, his parents, and two controls. The result was amazing! We thought then that the human genome contained about 100,000 L1 sequences, but now we know that the number is greater than 500,000. So it was a real surprise when Beth found that instead of a smear of thousands of L1 sequences, each individual had only a handful of bands and the patient had a new band not present in his parents (Figure 6). This band represented the insertion! Dombroski then obtained a commercial lambda genomic library and cloned four full-length L1s that hybridized with the JH-27 oligonucleotide and corresponded to four of the bands on the gel. Upon checking sequence of these four L1s, the second looked promising. With 5500 nts out of 6000 in hand, we had a perfect match with the insertion sequence (Figure 7). However, in those last 500 nts, Beth found two changes from the JH-27 insertion. When I saw those changes on the sequencing run, my first thought (optimistically) was that perhaps we had isolated an allele of the real precursor from the commercial library. To check that possibility, Beth got genomic sequence flanking the L1, did a PCR from the JH-27 oligonucleotide to the flank in both parents, and sequenced the region of the two changes from her PCR product. Voila! The parental sequences matched the insertion—the changes were not present in either parent and the allele hypothesis was probably correct. We then got blood from the mother, and Beth cloned the same L1 from the mother's genomic library. The mother's L1 was indeed identical in sequence over its last 3.8 kb to the insertion. It was also the first L1 isolated that contained two intact ORFs. It had originated on chromosome 22 and a portion of it had been copied and inserted into the factor VIII gene in JH-27. We had successfully obtained the precursor from among hundreds of thousands of L1s, and had strong evidence for cis preference of L1 elements. Then Abram Gabriel, an MD postdoc at Hopkins, suggested an assay for reverse transcriptase activity using a hybrid yeast Ty1-human L1 combination. Steve Mathias, a PhD student with Alan Scott, working with Abram and Jef Boeke, the yeast retrotransposon expert at Hopkins, demonstrated reverse transcriptase activity encoded by ORF2 and the two papers were published together.24Dombroski B.A. Mathias S.L. Nanthakumar E. Scott A.F. Kazazian Jr., H.H. Isolation of an active human transposable element.Science. 1991; 254: 1805-1808Crossref PubMed Scopus (348) Google Scholar, 25Mathias S.L. Scott A.F. Kazazian Jr., H.H. Boeke J.D. Gabriel A. Reverse transciptase encoded by a human transposable element.Science. 1991; 254: 1808-1810Crossref PubMed Scopus (547) Google Scholar All this time, I was learning more about transposable elements through quarterly lab meetings with Maxine Singer. In 1993, Susan Holmes, a grad student, worked up another interesting patient sample from the DNA diagnostic lab, a Duchenne patient with an L1 insertion knocking out his dystrophin gene. The insertion into an exon was unusual because it contained 500 nts of single-copy sequence 3′ to the L1 (Figure 8), which Susan used to clone the full-length precursor on chromosome 1q.26Holmes S.E. Dombroski B.A. Krebs C.M. Boehm C.D. Kazazian Jr., H.H. A new retrotransposable human L1 element from the LRE 2 locus on chromosome 1q produces a chimeric insertion.Nat. Genet. 1994; 7: 143-148Crossref PubMed Scopus (190) Google Scholar We know now that 20% of L1 insertions contain extra 3′ sequence because the L1 poly A signal is weak and cleavage of the L1 RNA often occurs only after a downstream poly A signal. Later, John Moran showed that this mechanism, called 3′ transduction, can be used by L1s to sh