msDNA and bacterial reverse transcriptase.

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Annu. Rev. Microbial. 1991. 45:J63-fl6 Copyright © 1991 by Annual Reviews lnc. All rights reserved

msDNA AND BACTERIAL REVERSE TRANSCRIPT ASE

Masayori Inouye and Sumiko Inouye Department of Biochemistry, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey at Rutgers, Piscataway, New Jersey 08854 KEY WORDS:

retroelements, mobile elements, retron

CONTENTS INTRODUCTION .................................................................................... .

164

msDNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

164 164 166 168 1 70 171 171 172 173 175 175 176 178 178 178 178 180 18 1 183 183

Discovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . General Features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genetic Locus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

BIOSYNTHESIS OF msDNA ..................................................................... . Requirement for Reverse Transcriptase . . . . . . . . . . . . . . . . . ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biosynthetic Pathway. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . General Features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . REVERSE TRANSCRIPTASE .................................................................... . Genetic Locus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

��;�U;�� ;�e;iff3;y:::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::

Codon Usage .................................................................................... . .

RETRON............................................................................................... . Structure and Distribution . . . . . . . . . . .. . . . . . . . ...... . . . . . . . . . . ..... . . . .... .... . . . . . . . . . . . . . . . . . . . Chromosomal Integration Site . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Phylogenetic Relationship and Origin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . . . . . Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

SUMMARY AND PROSPECTS .................................................................. .

163

0066-4227/91 / 100 1 -0 1 63$02.00

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INTRODUCTION In the fields of biology and biochemistry, unusual compounds of unknown function have often been discovered accidentally during the course of ex periments, and these compounds are later found to be very important for the living cell. Multicopy single-stranded DNA (msDNA) may be one such compound, although its functions have not yet been completely elucidated. This type of DNA was first found in Myxococcus xanthus, a gram-negative soil bacterium. The more recent discoveries of various msDNAs in Es cherichia coli and the requirement of reverse transcriptase (RT) for msDNA synthesis add an exciting dimension to this discovery. In particular, researchers have long believed that RT is specific to eukaryo tic organisms and does not exist in the prokaryotes (36, 37). Retroelements that encode RT have been identified in various forms, including retroviruses, retrotransposons, introns, and plasmids, and are widely dispersed among the eukaryotes such as mammals, insects, plants, and fungi (2). No compelling evidence has demonstrated the existence of retroelements in the prokaryotes, despite sporadic reports claiming the existence of RT activity in E. coli (1, 39) and in a halobacterium (20). However, bacterial RTs associated with msDNA biosynthesis are structurally related to retroviral RTs (7, 42). Thus, the existence of retroelements in the prokaryotes, the requirement of RT for msDNA synthesis, and the unusual structure-a 2' ,5' phosphodiester linkage between RNA and DNA of msDNA-offer formidable challenges for elucidating the evolutionary origin of retroelements, molecular mechanisms of msDNA biosynthesis, and functions and roles of msDNA in cells. Although a few review articles on msDNA ( 14, 22, 27, 36, 37) have been published, the present article provides a comprehensive review, emphasizing recent data on RT and the genetic element associated with it.

msDNA Discovery Myxobacteria are unique among bacteria in their ability to undergo multi cellular development. Upon starvation of nutrients, cells migrate by gliding to form multicellular aggregation centers, which then, in some species, develop into sophisticated fruiting bodies containing spores (32, 33). Because of these unique properties, myxobacteria have been used as a model system for studying developmental biology. During the course of experiments characterizing chromosomal DNA from two myxobacterial strains, Myxococcus xanthus and Stigmatella aurantiaca, a significant fraction of the DNA was found to reassociate with rapid, unimolecular kinetics, indicating the presence of large amounts of snap-

msDNA AND BACTERIAL RETROELEMENTS

165

back structures (44). However, an attempt failed to isolate this snapback material using an S 1 nuclease technique, which had been successfully employed to isolate rapid-renaturing structures from several enterobacteria


(31). The snapback material detected by optical reassociation analysis ex clusively resulted from low-molecular-weight extrachromosomal DNA. When the chromosomal DNA from M. xanthus was analyzed using elec trophoresis on a 5% acrylamide gel, a distinct satellite band was observed with a mobility corresponding to approximately 180 bp. This band was found to be a highly structured single-stranded DNA consisting of approximately 160 bases. This satellite DNA had 500-700 copies per genome in the M. xanthus cell. Thus, it was designated multicopy single-stranded DNA (msDNA). Similar msDNA was identified in S. aurantiaca (43) as well as in several other myxobacteria strains such as Myxococcus coralloides, Cystobacter virolaceus, Cystobacter ferrugineus, and Nannocystis exedens (4). Furthermore, msDNA was detected in nine independently isolated strains of M. xanthus as well as in Flexibacter elegans, a Cytophaga-like gliding bacterium distantly related to mxyobacteria. Although msDNA was not de tected in some myxobacterial strains such as Cystobacter fuscus and C. ferrugineus, the evidence suggested that msDNA may have originated in an ancestral myxobacterium (4). No msDNA was detected using ethidium bromide staining after polyacryla mide gel electrophoresis of DNA from an E. coli K12 strain, Edwardsiella

tarda, Salmonella typhimurium, Shigella dysenteriae, Serratia marcescens, Erwinia amylovora, Citrobacter freundi, Klebsiella aerogens, Proteus mirabilis, Pseudomonas aeruginosa, Neisseria gonorrhoeae, or Bacillus subtilis (43). Because of this result, many believed for a long time that E. coli does not contain msDNA. However, a recent discovery revealed that E. coli B does contain msDNA in contrast to E. coli K12 strains (26). Furthermore, 6% of clinical E. coli strains (23, 34), 13% of wild E. coli strains (10), and certain serotypes of E. coli (25) also contain msDNA. There fore, msDNA appears to be widely distributed among different bacterial species. The discovery of msDNA in E. coli B was a surprise and came serendipi tously, while Lim & Maas (26) were attempting to identify mRNA for an arginine biosynthetic enzyme by a primer extension. They detected a "cDNA" band even without the addition of a primer when a RNA preparation was used from E. coli B but not from strain KI2. They speculated that this cDNA band might have resulted from reverse transcriptase-catalyzed extension of the DNA strand of msDNA using the RNA molecule linked to the msDNA as a template. Indeed, they identified a specific msDNA molecule in E. coli B as described in the next section.

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Structure

As shown in Figure 1, the structure of six msDNAs are determined at present; three from myxobacteria and three from E. coli. These msDNAs are identified by two letters representing the host organism in which the msDNA is found and by the size of the single-stranded DNA (23). The determination of the msDNA structure took some effort and time because of its unexpected structure , a 2' ,5' phosphodiester linkage between RNA and DNA molecules. When msDNA was first discovered in M. xanthus (43), a RNA molecule was found attached at the 5 ' end of msDNA even after treatment with ribonucleases T l and A. A thorough study to determine the complete structure of the RNA molecule (called msdRNA) was carried out with msDNA from S. aurantiaca (8, 9) . This myxobacterium contains a msDNA of 163 bases that is highly homologous (81%) to msDNA-MxI62 from M. xanthus. The msDNA-SaI63 contains 2 1 base substitutions, 5 insertions, and 4 deletions when compared with msDNA-MxI62. However, almost all of these base substitutions are in the stem region of the DNA strand in such a way that the secondary structure is conserved (see Figure 1 ) . Researchers (8) isolated msDNA-SaI63 from S. aurantiaca cells treated with ribonucleases A and T l (RNaseA and RNaseT l , respectively) to remove RNA from the preparation. The remaining DNA was then labeled at the 5' end using T4 polynucleotide kinase and [r- 32p]ATP. However, the 5' label was unstable after treatment with 0 . 2 M NaOH, releasing an adenine ribonu cleoside 5' monophosphate. In addition, after this treatment, msDNA could no longer be labeled with kinase, indicating that rA is associated with, but not directly linked to, the 5' end of msDNA-Sa163. Thus, the investigators proposed that a branched RNA is linked to the 5' end of msDNA, which resembles the branch point of a lariat RNA , an intermediate structure formed during RNA splicing. Indeed, a triribonuc!eotide, 5' A-G-(C or U)3' was released when msDNA was treated with a debranching enzyme from HeLa cells (8). Because the G residue at the second position was identified only after the treatment with the debranching enzyme, the researchers concluded that the 5' -end residue of the single-stranded DNA of msDNA is branched out from the 2' -OH position of the G residue, fonning a 2',5' -phosphodiester

Figure 1 The proposed structures of various msDNAs from M. xanthus, S. aurantiaca, and E. coli. Boxes enclose msdRNA, and the branched rG residues are circled . The 5'-end RNA sequence of msDNA-Mx65 was determined as UGA through direct sequencing (5). However, on the basis of the facts discussed in the text, an additional 1 3-base sequence probably exists at the 5' end of the primary product of msDNA-Mx65. Similarly, the 5' -end RNA structure of msDNA-Ec73 is considered to have a sequence of 15 bases, which has not been determined by RNA sequencing (35). Other sequences cited are: msDNA-Mx I62 (6), msDNA-SaI63 (9), msDNA-Ec67 (23), and msDNA-Ec86 (26).

167

msDNA AND BACTERIAL RETROELEMENTS 't c a - 'G

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170

INOUYE & INOUYE

contains reverse transcriptase required for the synthesis of the msDNA (24) (see below). The 5' arms (the position of the RNA molecule upstream of the branched

G) of msDNA-Mx162 and msDNA-SaI63 are 20 and 19 bases in length, respectively (Figure 1). These sequences are a part of the sequences that form inverted repeats found in the msDNA coding region on the chromosome (see below). In the case of msDNA-SaI63 , however, this

5' arm has been shown


to be processed, or cleaved away, leaving a very stable msDNA with a 5' arm of only three bases

(9).

The fact that msDNA-Mx65 has a

5'

arm of three

bases (Figure 1) probably results from similar secondary processing of the RNA molecule. On the basis of the DNA sequence of the msDNA-coding region and the fact that the RNA molecule is processed at one base upstream of the a2 inverted repeat sequence (from msDNA-Ec67 and -Ec86; see the next section), the primary product of msDNA-Mx65 probably has a 5' arm of 16 bases

(5) (see the legend of Figure l ). By the same principle, msDNA 5' arm of 14 bases (Figure 1) (35).

Ec73 is considered to have a

Genetic Locus A DNA fragment coding for msDNA was first cloned for msDNA-Mx162 using the msDNA molecule labeled at the 5' end as a probe (43). The probe detected only one region of the

M.

xanthus chromosomal DNA that has the

sequence identical to the DNA sequence of msDNA. Later, the coding regions for msDNA msDNA-SaI63 (8,

(mst!)

and msdRNA

(msr) were cloned and sequenced for

9), msDNA-Mx65 (5), msDNA-Ec67 (23), msDNA-Ec86

(26), and msDNA-Ec73 (35). From these studies, one can make the following conclusions:

1. Only one locus resides on the bacterial chromosome for each msDNA. 2. Sequences identical to msDNA and msdRNA are found in this locus.

3. The coding region for msdRNA (msr) resides downstream of the coding region for msDNA

(msd) in such a way that these coding regions are in

opposite orientation, overlapping by 5 to 8 bases at their 3' ends (see the top position of Figure 2). 4. Within the sequence of

msd are a set of inverted repeats corresponding to (hI and b2 in Figure 2). In addition,

the stem-loop structure of msDNA

msDNA and msdRNA share another set of inverted repeats, al and a2; al

msd, and a2 is immediately upstream of the G residue within msr. As discussed in the next section, these

is immediately upstream of branched

inverted repeats are essential for msDNA biosynthesis because they allow the primary RNA transcript to form a stem structure (see Figure 2). The length and nucleotide sequence of the repeats are quite different for different msDNAs (Table

1).

171

msDNA AND BACTERIAL RETROELEMENTS -1lL-. .& ��:::: ::::::::� ==��====

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Figure

2 Biosynthetic pathway of msDNA synthesis. The retron region consisting of the msr-msd region and the gene for reverse transcriptase (RT) is shown on the top of the figure.

,

Solid arrows indicate the locations of two sets of inverted repeats (a I and a 2, and bl and b2). Open arrows indicate the genes for msdRNA

(msr) msDNA (msd), and RT. The

primary

transcript is considered to encompass the upstream region of msr through the RT gene, which is

step 1. The thick region in the RNA transcript corresponds to the final The branched G residue is circled, and the intiation codon for RT is also shown. On the

shown by a thin line at

msdRNA.

folded RNA. a triangle indicates the 5' -end processing site at the mismatching base. The dotted lines at steps 3 and

4 represent DNA strands. The figure is modified from one by Dhundale et al

(6) .

5. The promoter for the msr-msd region is upstream of msr, and transcription is from left to right, encompassing the entire region including the RT gene (see Figure 2 and the next section) . BIOSYNTHESIS OF msDNA

Requirement for Reverse Transcriptase The first indication suggesting that msDNA synthesis is quite different from chromosomal DNA synthesis came from labeling S. aurantiaca cells with [3H]-thymidine (9). In this system, msDNA-Sa163 synthesis was inhibited


1 72

INOUYE & INOUYE

by rifampicin (20 /Lg/mi) and chloramphenicol (100 /Lg/ml) but not by nalidixic acid ( l 00 /Lg/ml). These results indicate that msDNA requires rifampicin-sensitive RNA polymerase and a labile protein factor(s) but does not require DNA gyrase whose activity is known to be blocked by nalidixic acid. The same results were obtained for msDNA-MxI62 (6). How does the priming of DNA synthesis occur at the 2' OH of the G residue? If the primary transcriptional product from the msr-msd region is much longer at both the 5 ' and 3' ends than msdRNA itself, a partial duplex or stem structure may form between the sequences corresponding to the a l and a2 inverted repeats. The G residue at the end of the stem structure may serve as a primer for msDNA synthesis, which uses the same RNA transcript as a template for RT. S ynthesis of msDNA does indeed proceed according to this proposal . A primary product of the msdRNA for msDNA-MxI62 was identified by SI nuclease mapping to be approximately 375 bases in length, much longer at both 5' and 3' ends (6). Furthermore, a system utilizing permeabilized cells treated with phenethyl alcohol was established and r a-32PldCTP was in corporated into msDNA-Mx 1 62 (23) . In this study , intermediate structures were identified by interrupting the synthesis of msDNA-Mx162 with di deoxyribonucleotides. The [a-32P]dCTP-labeled products in the presence of ddGTP, ddATP, or ddTTP migrated during electrophoresis at the same position as the full-sized msDNA in acrylamide gels. However, treatment with ribonuclease A prior to gel electrophoresis resulted in many different sized bands, indicating that, during the labeling, intermediates are produced in which single-stranded DNAs of various lengths are associated with a compensatory length of RNA such that the total number of nucleotides for each intermediate is identical . These results provide clear evidence for msDNA synthesis by RT to support the model proposed by Dhundale et al (6). In addition, the results indicate a precise coupling mechanism of RT and ribonuclease H . A cell-free system was also established with use of a sonic extract of E. coli B cells to examine the synthesis of msDNA-Ec86 (26). Researchers have demonstrated that the in vitro DNA product is linked to a RNA, and, when ddTTP was used, all the chain termination products were larger than msDNA itself. After ribonuclease A treatment, a typical sequencing ladder appeared, indicating that the formation of the RNA-DNA linkage between msdRNA and msDNA is not a postsynthetic reaction but most likely the first step in msDNA synthesis. Biosynthetic Pathway

Figure 2 summarizes the biosynthetic pathway according to the results de scribed above. As discussed later, the primary transcript (pre-msdRNA) appears to contain an open reading frame for RT downstream of msr. This


173

transcript is probably processed between the msr msdregion and the RT ORF, producing a stable shorter RNA molecule, which is then folded by self annealing at step 2. The resulting stable structure serves not only as a primer but also as a template. The G residue that will form the branched linkage with msDNA resides at the very end of the stem formed by the al and a2 sequences in such a way that the G residue is readily accessible for priming msDNA synthesis. After the priming reaction at the 2 ' OH group of the G residue by a yet unknown mechanism, DNA synthesis proceeds using the same RNA molecule as a template (step 3). As DNA synthesis proceeds, the template RNA is processed by ribonuclease H activity until msDNA synthesis is completed (step 4).


-

General Features

The 5 ' end of pre-msdRNA for 75 bases upstream of msr (6). Thus, the promoter for the RNA transcript is at least 75 bp upstream of the msr locus. In the case of msDNA-Ec67 , the promoter ( - 1 0 and -35 regions) was assigned approximately 1 85 bp upstream of the msr locus (23). However, a recent primer-extension experiment indicates that the transcription starts much closer to the msr locus (M. Hsu, M. Inouye, & S. Inouye , unpublished results). On the other hand, msDNA-Ec86 from E. coli B could still be synthesized, even if the region upstream of msr was deleted up to residue - 1 4 (+ 1 for the first residue o f msdRNA) (26). S I nuclease mapping demon strated that the major transcript started at position + 1 , the first base of msdRNA, and included the msr-msd region as well as the downstream RT gene. Therefore , the promoter for msDNA-Ec86 is probably located very close to the msr locus. When the lacZ gene was fused within the RT gene for msDNA-Ec86, f3-galactosidase activities were 0 . 2 U and 1 .2 U for the deletion of the msr upstream region up to residue - 1 4 and the deletion up to residue - l 70 , respectively. These results suggest the possible existence of an enhancer sequence between - 170 and - 1 4 residues (26). The lacZ-RT fusion with a tac promoter fused at residue -14 produced 26.5 U of f3-galactosidase activity, indicating that the promoter for msDNA-Ec86 is extremely weak. INITIATION SITE OF THE RNA TRANSCRIPT

msDNA-MxI62 resides approximately

Intermediates detected in a cell-free system migrated at the same position as the full-sized RNA-linked msDNA in gel electrophoresis (see above) (2 1 ) . This indicates that pre-msdRNA is probably processed by an endo- or exonuclease, removing the upstream part of msdRNA at a very early stage of msDNA synthesis. An extra nucleotide resides at the 5 ' end of msdRNA in addition to the a2 sequence for msDNA-Ec67 (23) and msDNA Ec86 (26). For msDNA-SaI63 (9) and msDNA-Mx 1 62 (6), cleavage of the 5 ' end o f the RNA transcript appeared to occur at a base mismatch site within the a l -a2 stem structure, again leaving an extra nucleotide at the 5 ' end of RNA PROCESSING

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msdRNA. At the 3' end, ribonuclease H digests msdRNA base by base as DNA elongation proceeds along the RNA template (21 ) . RNA STRUCTURE NEAR THE BRANCHED rG RESIDUE The stem structure immediately upstream of the branched G residue is essential ( 1 2) . When three-base mismatches were introduced into the stem structure immediately upstream of the branched G residue, the synthesis of msDNA-Mx 1 62 was almost completely blocked (mutation A or B in Figure 3). However, if additional three-base substitutions were made on the other strand to resume the complementary base pairing, msDNA production was restored (double mutation A and B in Figure 3). Hsu et al ( 1 2) also found that the G residue could not be replaced with either C or A, while a dG residue could substitute

II

C

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I I I

l�

I I

CGUGGUGGUAGAAUGG-----

1/ I

wild-y t pe (+msDNA)

.......

-.....

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I I

C

C

AG GCA

C

U

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III

II I

-CGU U

CGUGGUGGUAGAAmutant (-msDNA)

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C AGGUAGAA-

muat nB t (-ms DNA)

C I C

--GCA AG

II I

�

III

- - C GU UCA GGUAGAAUGG ----.. double mutant (+ms DNA)

Figure 3

Mutations in pre-msdRNA. Only the region around the branched

rG residue for

msDNA-Mx162 is shown. The results are from Hsu et al (12). The branched rG residue is circled. Bold face indicates bases changed by mutagenesis, and an arrowhead indicates the conserved residue paired with the branched

rG residue.

G


1 75


for the 5' end (dC) of msDNA-Mx 1 62. In all these cases, the same amount of pre-msdRNA was produced. Interestingly, all msDNAs have an extra base between the stem structure and the residue complimentary to the 5' end of msDNA on the template strand. This extra base, which corresponds to the branched G residue, is always a G residue (arrowhead in Figure 3). TERMINATION O F msDNA SYNTHESIS The in vivo synthesis of msDNA terminates 49, 39, 49, 36, 57, and 55 bases before reaching the branched G residue for msDNA-Mx1 62, -Mx65, -Sa1 63, -Ec67, -Ec86, and -Ec73, respectively (see Figure 1) . Molecules of msDNA can serve as substrates in vitro for retroviral RTs to further extend the msDNA strand to the G residue once these molecules have been extracted from the cells and treated with phenol to remove proteins ( 1 0 , 23, 24, 26, 35). This precise termination does not appear to result from the inherent specificity of bacterial RTs because bacterial RT preparations were also able to extend the DNA strand when phenol-extracted msDNAs were used as substrates (24, 26). Furthermore, the RT for msDNA-Ec67 was found to be purified with msDNA-Ec67, which could serve as a substrate for the RT so that the RT could complete the DNA extension to the branched rG residue in vitro (24). These results indicate that a protein factor(s) may block the RT reaction at a very precise position in vivo before it reaches the branched G residue. Because the DNA fragments containing only the msr-msd region and the RT gene are sufficient for msDNA production in K 1 2 strains (23 , 26, 35), the gene for this protein factor does not seem to be associated with the genetic locus for the msDNA synthesizing system. This protein factor is probably bound to the stem-loop structure of msdRNA, which prevents RT from further extension. On the other hand, RT has been shown to be associated with the msDNA molecule in the cell, forming an approximately 1 9S complex in a glycerol gradient (23). Thus, one can assume that msDNA exists in the cell as a complex with RT and a RT-termination factor, and that the RT remains bound to the RNA-DNA hybrid of msDNA. One study (38) showed that msDNA Mx162 exists as a 14S complex with protein factors . This experiment showed that the sequence of msDNA from residue 60 to 1 0 1 is protected from dimethyl sulfate modification.

REVERSE TRANSCRIPTASE Genetic Locus

Dhundale et al (3) observed significant reduction in msDNA production in a deletion mutation at the region 1 00 bp upstream of the msd region for msDNA-Mx162 (in other words , downstream of the msr region) and an insertion mutation at a site 500 bp upstream of msd. These observations


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indicate that a cis- or trans-acting positive element required for msDNA synthesis is in this region . DNA sequencing led to the identification of an open reading frame (ORF) downstream of msr coding for a polypeptide of 485 amino acid residues, which showed sequence similarity with retroviral RTs (16). Similarly, an ORF for RT has been identified for all msDNAs shown in Figure 1 (see also Table 1) . The distances between the initiation site for msd and the ORF may vary from 19 to 77 bp, and the sizes of the RT ORF also vary from 316 to 5 86 residues (Table 1) . The RT ORF is essential for synthesis of msDNA-Ec67 (23), msDNA Ec86 (26), and msDNA-Ec73 (35). DNA fragments required for msDNA synthesis can be cloned into a K 1 2 strain of E. coli, reSUlting in production of msDNA in the K12 strain. These include fragments of 3.9 kb for msDNA Ec67 (23), 3.5 kb for msDNA-Ec86 (26), and 3.5 kb for msDNA-Ec73 (35 ) . A lacZ fusion with the R T ORF showed that the upstream msr-msd region and the downstream RT gene share the same promoter (23, 26). However, these two loci do not have to be in the same operon. If an appropriate promoter is added in front of a RT gene, the RT gene can be separated from the msr-msd region to support msDNA synthesis (35). In the case of msDNA-Ec73, an extra ORF (orj316) is in front of the RT ORF. These two overlap by four codons (35). The gene orf316 is dispensable for msDNA synthesis, and the RT ORF appears to have its own Shine Dalgarno sequence . Structural Diversity

The extensive size differences of the RT ORFs (Table 1) reflect the diversity of their domain structures. As summarized in Figure 4A, all bacterial RTs are quite different from eukaryotic RTs except for RT for msDNA-Ec67, which consists of a RT domain, a tether domain, and a RNase H domain. Both mxyobacterial RTs contain a large amino-tenninal domain of an unknown function, and the RTs for msDNA-Ec86 and -Ec73 consist of only the RT domain. Even within the RT domains, sequence similarities between the bacterial RTs are surprisingly few, as shown in Figure 4B . However, bacterial RTs share some common features indicating evolutionary relationships with retro viral RTs (see below) (42) . In particular, all the RT domains contain the YXDD sequence, the highly conserved sequence in all known RTs (boxed in Figure 4B). Interestingly, the X position is A in all bacterial RTs (see Figure 4B) . One finds within the RT domains of 250 to 260 residues 33 identical residues (solid circles in Figure 4B), out of which 1 1 residues are shared with eukaryotic RTs. The RT sequence alignment has many gaps. The two myx obacterial RTs appear to be more closely related. However, even within the RT domains, their identity is less than 50% . E. coli RTs do not show closer

msDNA AND B ACTERIAL RETROELEMENTS

A

RT

I

250-260

RNase H 120-130

177

I

Eukaryotic RT


msDNA·Mx162

: = = = = = = = = ••••••••C::I . -- ---____-=:1

msDNA·Mx65

�------

msDNA·Ec67

msDNA-Ec86

msDNA-Ec73

B

K K

R

R

MX162 Mx65

RWFAFHREVD TATHYVSWTI PKRDGS--KR TITSPKPELK AAQRWVLSNV VERLPVH--- -----GAAHG RHYSIIIRPRE RVRHYVTFAV PKRSGG--VR LLHAPKRRLK ALQRRMLALL VSKLPVS--- -----PQAHG

Be67

FLTNVLYRIG

E086

VETLRLLIYT ADFRYRIYTV EKKGPEKRMR TIYQPSRELK ALQGWVLRNI LDKLSSS--- -----PFSIG

95

Be73

TKGFASEVMR SPEPPKKWDI AKKKGG--MR TIYHPSSKVK LIQYWLMNNV FSKLPMH--- -----NAA¥A

73

MXl62

o G Y FVAGRSILTN ALA--HQGAD VVVKVDLKDF FPSVTWRRVK GLLRKGGLRE GTSTLLSLLS TEAPREAVQF FVPGRSIKTG AAP--HVGRR VVLKLDLKDF FPSVTFARVR GLLIALGYGY PVAATLAVUI TESERQPVEL

294 263

FERGKSIILN AYK--IIRGKQ

IILNIDLKDF FESFNFGRVR

GYFLSNQDFL

LNPVVATTLA ----------

154

FEKHQSILNN

FILNIDLEDF

GVFHSLGYNR

LISSVLT---

----------

1.50

FVKNRSIKSN ALLHAESKIIK YYVKIDLKDF FPSIKFTDFE YAFTRYRDRI EFTTEYDKEL LQLIKT----

139

SDNQYTQFTI PKKGKG--VR TISAPTDRLl{ OIQRRICDLL

•

•

•

•

SDCRDEIFAI RKISNNYSFG

•

226 195

96

A F

Mx65

Eo67 BeB6 Ee73

•

••

ATP--HIGAN

•

PQG MX162

FPSLTANKVF

•• •• • •

F Y DO

P

RGKLLHVAKG PRALPQGAPT SPGITNALCL KLDKRLSALA ---KRLGFTY TRYADDLTFS WTKAKQPKPR

361

Mx65

EGILFHVPVG PRVCVQGAPT SPALCNAVLL RLDRRLAGLA ---RRYGYTY TRYADDLTFS GDDVTA---

Ee67

326

-----KAACy NGTLPQGS:PC SPIISNLICN IHDMRLAKLA ---KKYGOTY RYAD IT TNKNTFPLEH -----KICCY KNLLPQGAPS SPKLANLICS KLDYRIQGYA ---GSRGLIY TRYADDLTLS AQSHKK---

216

-----ICFIS DSTLPIGFPT SPLIANFVAR ELDEKLTQKL NAIDKLNATY TRYADDIIVS TNMKGA----

200

e

Ee86

E 73

S

• •

••

G

•

•

K

•

D IS

•••••

•

208

LG

MX162

RTQRPFVAVL LSRVQEVVEA EGFRVHPDKT RVARK--GTR QRVTGLVV

MX65

407

-- --- -LERV RALAARYVQE EGFEVNREKT RVQRR--GGA QRVTGVTV

366

Eo67

ATVQPEGVVL GKVLVKEIEN SGFEINDSKT RLTYK--TSR QEVTGLTV

262

Eea6

------

RDFLFS!IPS EGLVINSKKT CISGP--RSQ RKVTGLVI

Eo73

248

-----SKLIL DCFKRTMKEI GPDFKINIKK FKICSASGGS IVVTGLKV

243

Figure 4

VVKA

•

•••

Comparison of bacterial RTs. (A) Domain structures of various bacterial RTs. The

the RT and RNase H domains, (8) Amino acid sequence alignment of bacterial RTs. Sequence alignment was carried out according to Xiong & Eickbush (42). Amino acids highly conserved in eukary ot ic RTs are shown on the top of the sequence. Amino acids conserved in five bacterial RTs are marked (0) . regions with closed bars and with stipled bars represent

respectively.

Numbers on the left indicate the amino acid positions from the amino terminus for each RT.

Sources for the sequences are: MX162 (16), Mx65 (15). Ec67 (23). Ec86 (26). and Ec73 (35).

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relatedness to any particular RTs. For example, RT-Ec73 shows 30, 27 , 26, and 25% identity to RT-MxI62, -Ec67, -Mx65, and -Ec86, respectively (35).


Enzymatic Specificity

Because DNA polymerase I has been shown to have intrinsic RT activity (28, 30), earlier claims for the existence of RT in bacteria remained rather ob scure ( 1 , 20, 39). Therefore, to identify the RT activity for msDNA-Ec67, a polA strain (DNA polymerase I-deficient) was used (23). In this experi ment, [a-32P]dGTP was incorporated when poly(rC)'oligo(dG) was used as a template-primer system. RT for msDNA-Ec86 was partially purified using column chromatography , and the RT preparation extended msDNA to the branched rG residue in the same manner as a retroviral RT (M -MuL V) (26). RT for msDNA-Ec67 was purified as a large-molecular-weight complex with msDNA (24). The complex sedimented in a glycerol gradient at an S value greater than 1 9 . The predominant protein species copurified with RT activity in the complex and had a molecular weight of 65 kilodaltons (kd), which is close to the expected size of 67,227 for RT-Ec67. The purified RT could produce single-stranded cDNA and double-stranded DNA as well, indicating that the bacterial RT can probably synthesize double-stranded DNA in vivo from mRNA like retroviral RTs.

Codon Usage

Table 2 shows codon usages of five bacterial RTs. One can see that codon biases are very similar to other genomic genes of M. xanthus for both myxobacterial RTs , while the codon usage of E. coli RTs are substantially different from that of other genomic genes of E. coli. These facts have very important implications for the evolution of these RTs as discussed in the next section. RETRON Structure and Distribution

Essential components required for msDNA synthesis can be confined to a short DNA sequence of 2 to 3 kb on the bacterial chromosome, although one cannot exclude the possible requirement of other cellular components. The msr-msd region and the RT gene belong to a single operon (see the previous section). One can consider this operon as a primitive retroelement, containing only a RT gene in contrast to other retroelements such as retroviruses [RT, integrase, long terminal repeats (LTR) , and virions], retrotransposons (RT , integrase, and LTR), retroposons (RT and integrase)' and pararetroviruses (RT, LTR, and virions). Thus, Temin (36) proposed the name retron to describe the element required for msDNA synthesis .

Table 2

Codon usage of the M _ xanthus and E _ coli reverse transcriptases M. Xanthus

--

Ala

Arg


a Mx162

Codon

aa

Asn As p eys G1n

Glu Gly

His Ile

Leu

1 17 3 20

9.3 5 1.2 3.1 36.4

15 0 9 3

6

eGU CGC CGA CGG AGA AGG

3 25 1 15 0 1

3 26 5 27 0 1

13. 0 64.1 0 15.2 3.3 4.3

3 1 3 4 13 7

4 2 2 2 8 4

I

GGU GGC GGA GGG

GAU GAC UGU UGC CAA CAG GAA GAG

eAU CAC AUU AUC AUA UUA UUG CUU eue eUA CUG

I I I I I

1 5 0 24 0 1 0

16

5 25 0

I

28

1 2

I

I

0

15

2

5

0

0 2 0 12

I I I I I

1 5 3 14 0 2 1

11

2

21

1

I

23

3 5

I

I

0

I I I I I

I

2

I

0 4 0

I

10

0 3 0 15 0

35

36

I

AM AAG

Met I

AUG

I

3

I

6

I

Phe

I

UUU uue

I

0 14

I

11

0

I

I

CCU CCC CCA CCG

I

Ly

s

Pro

Ser

UCU

uec UCA UCG AGU AGC

I

0 38

1

10 0

16 1

12 1

7

0

5

I

I

0 15

0

15

3 8 0

8 1 4

1

5

I

I

7.6 92.4

I

29 9

16.5 83.5

I I I I

34

0

100 3.6 96.4 15.7

84.3

16.2 75.2 4.3 4.3 14.3 85.7 16.3

81.3

2.5 0

7.3 0 33.9 1.8 56.9

5.8 94.2

2.9

97.1 12.9 37.1 0 50.0 1.3

40.0 0

20.0

1.3 37.3

7

8 3 10 2 19

7

9 4 13 6

I

8

Coli

2

5 2

I I I I I

9 7 9 1 4 1 5 3 10

4

7

I

3

6 3

I I I I I

I

4

12

I

5

I

I

28 4

I

11

I

I

9 1 7 2

5

9

I

11

5

4

2

13

I

I

12

10 4 12 5 3-

22 9

2

6 0 5 1 14

1 8

1

6 2

I I I I I

2 2

I

3

1.7

1.0

18 2

3

12

21.6

10 5

I

I

1

50.3 37.9 3.9 5.1

6

44 23

4 9

1 1 4 0 7 0

I

I

11

34.9

I

5 2

19

16 15 14 2 3 5

0

1

I

7

20.2 23.3

3

0

18

8 2

7

I

2

otherg Gene %

f EC73

Ec86e

EC67d

4 25 2 43

AAU AAC

I

Mx65b

GeU GCC GeA GeG

I I I I I

I

E. otherC Gene %

7

I

16

I

2

10

3 6 4 3 3

I

3; 1

I

9

I

;

I

I �I

32.3 67.7 54.2 45.8 42.1 57.9 28.0 72.0 71. 4

28.6

42.8

41. 1

6.2 10.0

44.3

55.7

41.3

55.1

3.6

8.5

10.1 8.2 9.5 2.4 61.3

76.0

24.0 -

43.7 56.3 12.5 7.4

17.6

62.5

6 0 9

20.4 8.9

2 4

9.8 27.5

2

20.5

12.9

1 80

INOUYE & INOUYE

Table 2


I

-

1

aa

(continued)

M.

Xanthus

-------

E. coli

-------

Codon

Thr

ACU ACC ACA ACG

Trp

UGG

9

Tyr

47.7 52.3

Val

31.8 17 .6 17.7 32.9

Total

a.a

485

427

586

320

316

"Inouye et al (16). bInouye et al (15).

Average from 5 M. xanthus genes (16). dLampson et al (23). o

eLim & Maas (26). 'Sun et al (35).

"Average from 199 E. coli genes (29).

All retrons so far identified exist at only one copy per chromosome. M. xanthus, however, has two independent retrons, retron-Mx l 62 (6, 1 6) and retron-Mx65 ( 1 5). In contrast to M. xanthus, a minor population of E. coli strains contains retrons as judged by the ability to produce msDNA. The fact that msDNA is produced in E. coli B but not Kl2 (26) suggests that some other E. coli strains may produce msDNA. When 1 13 clinical isolates of E. coli were tested for msDNA, 7 strains were found to contain msDNA (34). Of these strains, two have been characterized so far, one for retron-Ec67 (23) and the other for retron-Ec73 (35). Distribution of retrons in E. coli strains is not restricted to clinical strains. Strains isolated from healthy people as well as wild strains isolated from their natural habitats contain msDNA ( 1 0). Chromosomal Integration Site

Because of the extensive diversity of retrons in E. coli, it is interesting to know where and how retrons are integrated into the E. coli chromosome. Retron-Ec67 was m apped at a position equivalent to 19 min of the K 1 2 chromosome ( 1 1 ). The element containing the retron consisted of a unique 34-kb sequence that was flanked by 26-bp direct repeats. This observation suggests that the 34-kb foreign DNA fragment containing retron-Ec67 was



181

integrated into the E. coli genome as a movable element. Interestingly, the 34-kb fragment contained an ORF of 285 residues that has 44% identity with the E. coli Dam methylase. Retron-Ec73 was found to be a part of a 1 2 . 7-kb foreign DNA fragment flanked by 29-bp direct repeats and integrated into the gene for selenocystyl tRNA (selC) at 82 min on the K 1 2 chromosome (35) . Except for the 2 . 4-kb retron region, the foreign DNA fragment showed remarkable sequence sim ilarity to most of the bacteriophage P4. Among the phage genes, however, the integrase gene had rather low identity (40%) to P4 integrase. This cryptic prophage was excised from the host chromosome when phage P2 infected the host strain (17). Phage P4 is known to require a helper genome such as phage P2 to provide the late gene functions for lytic growth . The excised prophage genome was then packaged into an infectious virion. The newly formed phage designated retronphage 4JR73 closely resembles P4 as a virion and in its lytic growth. Most importantly , retronphage 4JR73 could also lysogenize a new host strain, reintegrating its genome into the selC gene of the host chromo some and enabling the newly formed lysogen to produce msDNA-Ec73 . Thus retron-Ec73 is associated with a mobile element and can be transferred from cell to cell. Some ORFs in the 34-kb foreign fragment containing retron-Ec67 are similar to genes of phage P I 86 (M. Hsu, M . Inouye, & S . Inouye, un published data) , suggesting that this fragment may also be a cryptic prophage. Retron-Ec86 appears to be integrated at 19 min on the E. coli B chromosome (D . Lim & W. Maas, personal communication). Phylogenetic Relationship and Origin One should note the distinct differences between myxobacterial retrons and E. coli retrons . First, retron-Mx l 62 is found in all natural isolates of M. xanthus so far examined. Twenty strains isolated throughout the world (New York, California, Fiji Island, Spain, Italy, Poland, France, Germany, Turkey, and Greece) were tested for the presence of rnsDNA-Mx I62 and the gene for RT-Mx 1 62 using Southern blot analysis (B . C. Lampson, M . Inouye, & S . Inouye, unpublished results) . Without exception , all strains contain retron Mx 1 62 . In addition, S. aurantiaca , a different species of myxobacteria, also contains a highly homologous msDNA, msDNA-SaI 63 (9) (see Figure I ) . Most o f the base substitutions between msDNA-Mx 162 and -Sa163 occurred such that the stable secondary structure in msDNA is maintained . These facts clearly suggest that both retron-Mx l 62 and -Sa163 share a common pro genitor retron, which existed in a common ancestral bacterium prior to the divergence of the two myxobacterial species . The fact that the codon usage of


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RT-Mx 162 is almost identical to those found in other M. xanthus genes (Table 2) supports the notion that the M. xanthus RT gene is as old as other genomic genes (15). The mxyobacteria are believed to have diverged from their nearest bacterial relatives about 2 x 109 years ago ( 1 9). This belief leads to the argument that myxobacterial RTs are ancient and existed before eukaryotes evolved ( 1 6) . In contrast, only a minor population of wild E. coli strains ( 1 3%) ( t o) contains retrons , and these retrons are extensively different from each other with regard to their msDNA and RT structures 00, 23 , 26, 35). These facts indicate that E. coli retrons were more recently acquired from some foreign sources that remain to be identified. Xiong & Eickbush (42) have constructed a phylogenetic tree of 82 different RTs from animals, plants , protozoans , fungi, and bacteria. From this tree, they proposed that retrotransposons are most likely progenitors of all current retroelements , from which the LTR branch and the non-LTR branch were derived . They also suggested that non-LTR retrotransposons are the oldest group of retroelements , and bacterial RTs were captured from the non-LTR retrotransposons later during evolution . This hypothesis conflicts with the proposal above in which the mxyobacterial RTs are the oldest. This discrep ancy is considered to stem from the assumption in the latter hypothesis that all the retroelements are subject to an extremely error-prone system arising from the poor fidelity of DNA synthesis by RT (14). However, the RT genes in myxobacteria are part of the chromosomal DNA and are replicated not by their own gene product (RT), but rather by a DNA-dependent · DNA polymerase of high fidelity. Therefore, the mutations in the contemporary myxobacterial RT genes are considered to have accumulated at far slower rates than those for eukaryotic RT genes , which have been replicated by highly error-prone RTs. If this consideration is taken into account, a sub stantially different phylogenetic tree could be established. A puzzling fact, however, is that E. coli msDNAs and RTs are widely divergent, suggesting that before the integration of the retrons into the E. coli genome , they were replicated by a highly error-prone system. Thus, it is tempting to speculate that RTs were used for the retron' s reproduction in that system. The recent discovery of retronphage cpR73 may help provide an answer to this question ( 1 7 , 35). Although retronphage cpR73 has its own DNA primase gene, the phage genome may be replicated under certain circumstances by its own RT using selenocystyl tRNA and/or msDNA-Ec73 as primers (17). If such an event happens in the life cycle of retronphage cpR73 , many mutations would accumulate in the gene for RT-Ec73 . An alternative hypothesis proposed that E. coli retrons might have ancient ori gins, having existed in other organisms before their relatively recent (in terms


1 83

of evolutionary time) transfer to certain E. coli strains by several independent events ( 14) .


Function

The fact that for all retrons so far characterized there is only one per genome suggests that they are not selfish DNA. Furthermore , the fact that all natural isolates of M. xanthus contained retron-Mx l 62 indicates that cells with the retron have some selective advantage in natural habitats over cells without the retron. However, under laboratory conditions, no phylogenetic differences were detected between the wild-type strain and a mutant strain that could not produce both msDNA-Mx I62 and -Mx65 (5). In all msDNA molecules examined (Figure I), RT does not elongate msDNA all the way to the branched G residue , thus leaving unused template RNAs of 40 to 80 bases in length. The sites of this blockage are very precise for all msDNA molecules, but the blockage is eliminated if msDNA mole cules are treated with phenol to remove protein components . These facts suggest that the 3 ' -end structure of msDNA may be important for its function. Multicopy single-stranded DNA may serve as a primer for cDNA synthesis from a specific mRNA template if the mRNA contains a sequence com plementary to the 3 ' -end sequence of the msDNA. In M. xanthus, such an event might happen in a specific stage of cellular differentiation of the bacterium.

SUMMARY AND PROSPECTS

During the past seven years since the discovery of msDNA, a substantial amount of knowledge has accumulated concerning its structure and biosynth esis. However, as we learn more about msDNA, more questions seem to appear. Some of the major questions can be summarized as follows: 1. How is DNA synthesis primed at the 2 ' OH group of the rG residue? Bacterial RTs use their own template RNAs as primers, while retroviral RTs require cellular tRNAs as primers for the initiation of DNA synthesis. Such a self-priming function of a template RNA appears to be an attractive feature for a primitive RT or primitive DNA synthesis. Therefore , to know whether the priming reaction is enzymatic or nonenzymatic is important. Recently. a debranching enzyme was found to be required for efficient in vivo transposi tion of yeast Ty l elements (K. B . Chapman & J. D. Boeke, personal communication), This observation may indicate that DNA initiation for Ty l elements occurs at a 2 ' OH position; failure to debranch this linkage may reduce transposition efficiency,


1 84

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2 . How widely is the 2' OH priming reaction used? And how widely are retrons and RTs distributed, not only in other prokaryotes besides myxobac teria and E. coli, but also in archaebacteria and eukaryotes? The discovery of RT in the prokaryotes raises an important question with regard to the origin of RT, particularly if the RNA world preceded our current DNA world at a very early stage of life (18, 40) . If earlier genomes were composed of RNA, RT could have played an essential role in their conversion to DNA. The bacterial RT' s associated retrons may be closely related to the original RT. 3. Why are msDNA and bacterial RTs so diverse? Although the retrons found so far are integrated into bacterial genomes and replicated by DNA polymerase but not by RT, they are considered to have existed in highly error-prone systems before they settled in the bacterial genomes. What are these systems? The recent discovery of retronphage

The reverse transcriptase.

Antiretroviral therapy: reverse transcriptase inhibition.

Telomerase reverse transcriptase regulates microRNAs.

Reverse transcriptase and intron number evolution.

The non-reverse transcriptase activity of the human telomerase reverse transcriptase promotes tumor progression (review).

Reverse transcriptase from avian myeloblastosis virus.

Specific HIV-1 reverse transcriptase inhibitors.

HIV inhibitors targeted at the reverse transcriptase.

NMR characterization of HIV-1 reverse transcriptase binding to various non-nucleoside reverse transcriptase inhibitors with different activities.

Characterisation of visna virus reverse transcriptase: a micro scale reverse transcriptase assay adapted for use with an automated cell harvester.

HIV-1 Protease, Reverse Transcriptase, and Integrase Variation.

Rapid screening of HIV reverse transcriptase and integrase inhibitors.

Purification, characterization and crystallization of recombinant HIV-1 reverse transcriptase.

Absence of telomerase reverse transcriptase promoter mutations in neuroblastoma.

Asymmetric conformational maturation of HIV-1 reverse transcriptase.

Human Specific Regulation of the Telomerase Reverse Transcriptase Gene.

Regulation of the Telomerase Reverse Transcriptase Subunit through Epigenetic Mechanisms.

Occurrence of particle-bound reverse transcriptase in human amniotic fluid.

Arylsulfone-based HIV-1 non-nucleoside reverse transcriptase inhibitors.

HIV-1 reverse transcriptase inhibitor from Phyllanthus niruri.

Transcriptional Regulation of Telomerase Reverse Transcriptase (TERT) by MYC.

Sodium pyrophosphate inhibition of RNA.DNA hybrid degradation by reverse transcriptase.

Characterization of reverse transcriptase from filine leukemia virus by radioimmunoassay.

Quantitation of avian RNA tumor virus reverse transcriptase by radioimmunoassay.