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15 Portier, C., Dondon, L., GrunbergManago, M. and Regnier, P. (1987) EMBOJ. 6, 2165-2170 16 Melefors, O. and von Gabain, A. (1988) Cell 52,893-901 17 Uzan, M., Favre, R. and Brody, E. (1988) Proc. Nat] Acad. Sci. USA 85, 8895-8899 18 Ross, J., Peltz, S. W., Kobs, G. and Brewer, G. (1986) Mol. Cell. Biol. 6, 4362-4373 19 Ross, J. and Kobs, G. (1986) Mol. Cell. Biol. 6, 579-593 20 Shaw, G. and Kamen, R. (1986) Cell 46, 659-667 21 Caput, D., Beutler, B., Hartog, K., Thayer, R., Brown-Shimer, S. and Cerami, A. (1986) Proc. Natl Acad. Sci. USA 83, 1670-1674 22 Wilson, T. and Treisman, R. (1988) Nature 336, 396-399 23 Jones, T. R. and Cole, M. D. (1988) Mol. Cell. Biol. 7, 4513-4521 24 Schuler, G. D. and Cole, M. D. (1988) Cell 55, 1115-1122 25 Kabnick, K. S. and I-Iousman, D. E. (1988) Mol. Cell. Biol. 8, 3244-3250 26 Shyu, A-B., Greenberg, M. E. and Be]asco, J. G. (1989) Genes Dev. 3, 60-72 []

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The in vivo application of ribozymes Matt Cotten The ability to inhibit the e x p r e s s i o n of a specific gene selectively has at least two c o m p e l l i n g applications. • As a tool for m o l e c u l a r biology. T h e n u m b e r of genes that have b e e n c l o n e d and s e q u e n c e d has risen exp o n e n t i a l l y and further s t u d y of the functioning of these genes has necessitated the d e v e l o p m e n t of a simple and r e p r o d u c i b l e t e c h n i q u e that switches e x p r e s s i o n of the gene o f f the effect this has on the cell can t h e n be analysed. • For generating genetically modified organisms. Virus-resistant plants a n d animals, novel strains of organisms, or plants and animals that no longer possess a certain undesirable trait can be generated if the capacity to block gene e x p r e s s i o n selectively exists. In the past decade, the c o n c e p t of ~) 1990, Elsevier Science Publishers Ltd (UK)

using an antisense nucleic acid seq u e n c e to h y b r i d i z e with, and block the function of, a m e s s e n g e r RNA b e c a m e p o p u l a r (see Ref. 1 for a review). The i n h i b i t o r y action of antisense m o l e c u l e s on gene expression d e p e n d s either on the stability of the antisense-RNA-target-RNA h y b r i d or on the a n t i s e n s e - D N A RNA h y b r i d triggering a p r o t e i n e n z y m a t i c d e s t r u c t i o n of the target RNA. However, more recently, exp e r i m e n t s d e m o n s t r a t e d that RNA itself could have enzymatic activity 2'3 and, more importantly, that these catalytic RNA molecules (ribozymes) c o u l d be m o d i f i e d to create antisense RNA units that b o t h b i n d to target RNA m o l e c u l e s and catalyse their cleavage 4-6. The use of the catalytic RNAs has been a d o p t e d rapidly in the h o p e that t h e y c o u l d be used w i t h i n cells to bind, cleave and block the activity of specific

0167 - 9430/90/$2.00

Cell. Biol. 9, 1996-2006 39 Fort, P., Rech, J., Vie, A., Piechaczyk, M., Bonnieu, A., Jeanteur, P. and Blanchard, J. M. (1987) Nucleic Acids Res. 15, 5657-5667 40 Rahmsdorf, H. J., Schontahl, A., Angel, P., Liftin, M., Ruther, U. and Herrlich, P. (1987) Nucleic Acids Res. 15, 1643-1659 41 Huez, G., Marbaix, G., Burny, A., Hubert, E., Leclercq, M., Cleuter, Y., Chantrenne, H., Soreq, H. and Littauer, U. Z. (1977) Nature 266, 473-474 42 Graves, R. A., Pandey, N. B., Chodchoy, N. and Marzluff, W. F. (1987) Cell 48,616-626 43 Bernstein, P., Peltz, S. W. and Ross, J. (1989) Mo]. Cell. Biol. 9, 659-670 44 Grossi de Sa, M. F., Standart, N., Martins de Sa, C., Akhayat, Oo, Huesca, M. and Scherrer, K. (1988) Eur. J. Biochem. 176, 521-526 45 Kruys, V., Marinx, O., Shaw, G., Deschamps, J. and Huez, G. (1989) Science 245,852-855 46 Tonouchi, N., Miwa, K., Karasuyama, H. and Matsui, H. (1989) Biochem. Biophys. Res. Commun. 163, 1056-1062

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target RNA molecules, possibly proving to be more effective inhibitors t h a n antisense RNA or DNA. Currently, the most p r o m i s i n g r i b o z y m e for targeted in vivo use is the h a m m e r h e a d r i b o z y m e , a ribozyme that contains the catalytic d o m a i n f o u n d in several plant viroid RNAs and that m a y be m o d i f i e d using flanking sequences to position the catalytic d o m a i n at any c h o s e n target site (Fig. 1; Ref. 6; see also Ref. 7 for a concise r e v i e w of the developm e n t of the h a m m e r h e a d m o t i f into a targeted ribozyme). If the targeted h a m m e r h e a d r i b o z y m e can f u n c t i o n efficiently to destroy its target RNA in the p r e s e n c e of p r o t e i n and, more importantly, if it can f u n c t i o n w i t h i n a living cell, it s h o u l d be possible to cleave selectively, and t h e r e b y inactivate, any RNA molecule. Molecular biologists are n o w beginning to test these m o l e c u l e s against intracellular targets. T h e r e are, h o w e v e r , a n u m b e r of questions c o n c e r n i n g essential p r o p e r t i e s of the r i b o z y m e yet to be answered. H o w efficiently do targeted ribozymes f u n c t i o n to cleave target RNA m o l e c u l e s w i t h i n living cells? What is the best way to deliver these

TIBTECH - JULY 1990 [Vol. 8]

~Fig.

175

1

cleavage site I

F

5' AAGUGUUACAGCUCUUUUAGAAUUUGUCUAGCAGG...3' CACAAUGUCGA AAAAUCUUAAAC 5' targeting A m

i

n

i

i

i

i

=

i

m

n

A

G

C G

i

i

n

n

n

m

m

i

n

CUG A

AGU

A U

G C G C A G G U

catalytic

i

n

targetRNA

u

sequences I Ribozyme

domain

The hammerhead ribozyme. Based on the model of Haseloff and Gerlach 6, a hammerhead ribozyme was designed to cleave a U7 RNA target 29 The hammerhead ribozyme shown here contains the 22 nucleotide (mildly hammerhead shaped) catalytic RNA sequence from the satellite tobacco ringspot virus (catalytic domain, indicated in bold letters) flanked by 11 and 12 nucleotide designer targeting sequences that serve to position the catalytic domain at the target site by Watson-Crick base pairing. Cleavage of the target RNA occurs after the indicated CUC residue, generating a 2 ' 3 ' cyclic phosphate and a 5' hydroxyl. The targeting sequences can be varied to suit the targeting requirements; so far there has been no demonstration of a RNA sequence that interferes with cleavage activity. The wild-type viral cleavage sites contain the trinucleotide GUC, however, mutational analyses have demonstrated that other trinucleotides (especially GUU, GUA, CUC and UUC) can function at this position provided that the targeting sequence is altered to maintain the appropriate base pairing 43

ribozymes into the cell? And most importantly, does the use of ribozymes offer any advantages over antisense RNA (reviewed in Refs 1 and 8) and antisense DNA oligonucleotides (reviewed in Refs 9 and 10) for the inhibition of gene expression? A number of research groups are actively pursuing the answers to these questions. In this article I present some of the strategies that are being used and some of the initial answers.

Introduction of ribozymes into cells There are two general approaches that can be used to introduce ribozymes into cells, each with its own limitations and advantages. The first approach involves synthesis of the ribozyme molecule in vitro and introduction of the ribozyme RNA from outside. The second strategy involves creating ribozyme-encoding genes and introducing these DNA elements into cells so that the cell itself produces the ribozymes. Inhibition by ribozymes from without Using ribozymes generated & vitro allows considerable flexibility in the

method of their synthesis. The bacteriophage RNA potymerase systems and a cloned or synthetic oligonucleotide template may be used to generate the RNA in vitro. Protected ribonucleotide building blocks are also available, allowing the automated chemical synthesis of ribozymes. The chemical approach may permit modifications that increase the stability of the ribozyme or improve the ribozyme's catalytic activity. The next question is h o w to get these ribozymes into the cell. The cell membrane presents a substantial barrier to the entry of highly charged, high molecular mass compounds. Recently, a naturally occurring receptor that may be involved in the transport of short oligonucleotides has been described. However, this transport mechanism appears to have a size limit in the range of 20 nucleotides, limiting its usefulness for the delivery of the ribozyme molecules (which are approximately 50 nucleotides) 11. There have been several reports of RNA introduction into cells using recently developed transfection techniques such as the DOTMA cationic liposome-mediated

transfection 12,13, electroporation 14, microinjection 15 or calcium phosphate precipitation 16 and direct injection of nucleic acids into muscle tissue 17. These techniques should also function to deliver ribozymic RNA to cells. Will these approaches deliver enough functional ribozyme for successful inhibition? A rough calculation based on the values for DOTMA reported by Malone eta]. 12 suggests that 1-3 ~g RNA is 'associated' with ten million cells, This corresponds to a delivery of approximately one million molecules per cell, for a 50 nucleotide ribozyme. If this number represents intracellular, active, undegraded ribozyme and, more importantly, represents molecules within the same cellular compartment as the target RNA, this approach looks promising. Microinjection by-passes the uptake problem and will be very useful for testing the intracellular behavior of ribozymes (especially with the automated microinjection system n o w available ~8) but it does limit the number of cells that can be processed. One drawback with DOTMA and electroporation is the harsh effect of these treatments on cells. When working on a particularly delicate and easily triggered cellular event, the use of these reagents can obscure the biological process being studied. Some of the commonly used techniques, such as DOTMA, electroporation and microprojectile transfection, use the strategy of disrupting the cell membrane briefly to gain entry. To develop a gentle, efficient method of introducing nucleic acids such as ribozymes into cells we have chosen instead to use one of the 'revolving doors' that the cell possesses. The approach, based on the work of Wu and Wu 19'20, involves pirating a natural endocytotic event to deliver the compounds to the cell. Eukaryotic cells require iron, most of which they obtain bound to the iron transport protein, transferrin. This protein is rapidly endocytosed by cells after binding to one of the abundant transferrin receptors present on the cell surface. We modified the iron transport protein transferrin with polycationic domains to convert transferrin into a nucleic acidbinding protein 21-23. The binding and internalization of transferrinpolylysine complexes can be used to

176

transport nucleic acids into the cell. Although many of the initial studies have used these molecules to deliver large, gene-sized DNA molecules into cells, our preliminary experiments suggest that these conjugates are also useful for the delivery of short, single-stranded nucleic acids such as ribozymes. One major consideration is that approaches involving externallysupplied ribozymes produce only a transient effect: once the introduced ribozyme RNA has been degraded, the party is over. A number of studies demonstrate that antisense DNA oligonucleotides with chemical modifications that increase the molecule's stability in vivo are more potent inhibitors than unmodified oligonucleotides (reviewed in Ref. 9). Perhaps similar modifications may be adopted to enhance the stability of chemically synthesized ribozymes (provided chemical modifications that do not interfere with the catalytic activity of the ribozyme can be identified).

Inhibition by ribozymes from within A second method of applying ribozymes is to insert ribozyme 'genes' into target cells. The size of the molecule inserted will, of course, be significantly larger than the sequence required just to encode the ribozyme. A ribozyme 'gene' must contain both the DNA encoding the ribozyme itself a n d the DNA signals that tell the cell to transcribe the information into RNA. These genes would range from a 1000-5000 bp polymerase II gene containing promoter/enhancer elements, introns and 3' polyadenylation signals for conferring stability on the transcript, to the more compact polymerase III genes of less than 200 bp in size. As with the direct ribozyme approach, the main problem is introducing the nucleic acid into the target cell. However, there are two major advantages to this approach: first, the inserted ribozyme gene (and therefore the ability to produce ribozymes) will be inherited by all the cell's progeny, and second, the insertion of just one ribozyme gene can result in the production of a large number of ribozyme molecules. Furthermore, the gene will continue to be transcribed to produce ribozymes, the effect is not transient. Such features are important considerations for the biotechnological

TIBTECH - JULY 1990 [Vol. 8]

--Box I

Ribozyme expression strategies a

initiation site Strong promoter

--terminator

i.

I s00- 2000 bp 1 transcription _

5'CAP

[ " .... ~--=i ...... ]n

AAAAAAA ...

b i) a tRNA gene initiation site

t~rmln~fnr transcription

,, ', i=

,, [ approx. 100 bp I

ii) a ribtRNA gene initiation site

cleavage target site

~ RNA

terminator

,

transcription

11|1111

|~1|11|

t

1

I"

t

=-i

I approx.

150bp I

=i

ribozym~~

(a) A polymerase II expression strategy. A gene is created containing the upstream DNA signals for strong RNA potymerase II expression (frequently a viral element or a retroviral long terminal repeat, but occasionally a strong endogenous promoter element such as an actin gene promoter is used) 25. The DNA encoding the ribozyme is inserted downstream from this sequence so that the resulting transcript contains an active ribozyme. High level expression in eukaryotic cells requires the presence of a polyadenylation site at the 3' end of the gene 44. This DNA sequence may function to tell the RNA polymerase to stop synthesis but certainly serves as a signal for the cleavage of the nascent RNA molecule and the attachment of approximately 200 adenosine nucleotides (poly A) to the transcript. This poly A tail serves to stabilize the RNA against degradation and may serve other functions. The presence of an intron sequence on the transcript (routing the transcript through the normal RNA splicing machinery) may increase the stability or ensure the nuclearcytoplasmic transport of RNA molecules. During the transcription process, a cap structure is synthesized at the 5' terminus; this structure may stabilize the 5' end and functions during translation initiation. (b) A polymerase III expression strategy. A transfer RNA gene. In contrast to polymerase II transcription units, the promoter elements (the A box and B box) for polymerase III genes are found within the gene, the same sequences are then found in the resulting RNA molecule (reviewed in Ref. 26). The transcriptional terminator is a stretch of four or more thymidine residues. The ribozyme-encoding DNA is inserted between the A and B box; transcription of this unit produces a compact tRNA molecule with a ribozyme embedded in the anticodon loop.

production of transgenic organisms. Such a system, using ribozymeencoding sequences embedded in the 3'-untranslated region of a po]-

ymerase II transcript driven by an SV40 early promoter, has been successfully used to generate a 30-60% inhibition of a chloramphenicol acetyl-

TIBTECH - JULY 1990 [Vol. 8]

transferase activity in mammalian cells 24. This polymerase II system requires a large (1000-fold) molar excess of the ribozyme over target RNA for the inhibition effect. The polymerase II strategy has also been successfully used to block replication of the AIDS virus, HIV-1, in a tissue culture system 25. A strong human actin promoter was used to drive transcription of a single ribozyme element targeted to the viral gag gene. A PCR-based RNA analysis detected RNA molecules consistent with cleavage of the target RNA. A strategy to increase the yield of ribozyme per pol II gene takes advantage of the large coding capacity of the po] II gene. It is possible to create a multimeric ribozyme gene with interspersed, self-cleavage sequences. A single transcript could contain 10-20 ribozymes, and selfprocessing of this precursor would therefore yield a large number of ribozyme molecules. This approach may require modification, such as the addition of RNA secondary structure, to increase the stability of the monomeric ribozymes once they are released from the protection afforded by the 5' cap structure and the 3' polyadenosine elements of the multimeric precursor RNA. We chose to use a polymerase III gene to generate intracellular ribozyme molecules. In general, these genes are expressed at an order of magnitude higher than polymerase II genes and appear to be expressed in all tissues (reviewed in Ref. 26). Polymerase III genes include the genes that give rise to tRNA molecules, to 5S RNA molecules and to the U6 RNA. Earlier studies have shown the usefulness of the adenovirus VA1 gene, also transcribed by polymerase III, to generate antisense RNA molecules 27. The gene that we use is a methionine tRNA gene from either X e n o p u s ]aevis or humans. By inserting the DNA encoding a hammerhead ribozyme into the anticodon loop region of the gene, a 190 bp gene, that is transcriptionally very active and produces a 125 nucleotide 'ribtRNA' molecule (Fig. 2b) was created. This molecule (as synthesized in the Xenopus oocyte) is functional as a ribozyme both in vitro and in vivo 28. The small size of the po] III ribtRNA genetic unit (less than 200 bp) permits multiple copies of ribozyme

177

genes to be included on a single bacterial plasmid, thereby increasing the gene dose per plasmid. Furthermore, because not all target sites may be equally accessible, we can assemble vectors containing a cocktail of ribtRNA genes aimed at multiple sites in target RNAs. This increases the likelihood of successful targeting. Molecular biologists have relied upon strong po] II viral enhancer/promoter elements to ensure high expression of engineered genes. There is always the danger, however, that the chromosomal integration of these elements, especially the retroviral LTRs, may activate nearby, cellular genes resulting in the inappropriate expression of these genes. Since this very same event is implicated in a number of types of cancer, the use of these sequence elements may be hazardous. The po] III gene, in contrast, has no demonstrated cis enhancer activity and contains a very strong transcriptional terminator (a string of four or more T residues); the likelihood of inappropriate activation of adjacent genes is therefore minimized.

understood involves the excess of ribozyme (or antisense RNA molecule) required for successful inhibition. Two different studies have shown a requirement for a high (1000 : 1) ribozyme:substrate ratio for inhibition in vivo in eukaryotic cells 24'28. This suggests that the catalytic potential of the ribozyme is not being achieved. On the other hand, no direct comparison with antisense RNA or DNA has been performed in vivo to determine if the cleavage capacity of the ribozyme is involved in the observed inhibition (although the observation by Sarver et al. 25 of the expected cleavage products suggests that cleavage is involved). The hammerhead ribozyme has been shown to work in prokaryotic cells when the ribozyme RNA is part of the same RNA molecule as the target RNA. No effect was seen when the ribozyme and target RNA molecules were generated from different genes 32. Therefore, when co-localization within the cell is maintained (with the two components, ribozyme and target, on the same molecule) the ribozyme can function at a 1 : 1 ribozyme :target ratio. It is now being realized that What limits ribozyme activity? t h e r e i s a highly organized spatial One of the limits to ribozyme distribution of RNA molecules within cleavage activity may be the accessi- the cytoplasm and the nucleus (see, bility of the target site. When testing for example, Ref. 33). Thus, merely ribozymes against the snRNA U7, a insuring high-level expression of the snRNA required for histone mRNA ribozyme may not be sufficient for synthesis, we tested both a GUC and ribozyme success. We need to ensure a CUC target in the RNA molecule. high ribozyme expression within the While both ribozyme target sites were same compartment of the cell occuequally cleavable in the absence of pied by the target RNA. protein, we found that only the CUC site could be cleaved by the ribozyme Alternative ribozymes in the presence of the naturally The considerable target-site flexibound proteins. Digestion of the RNP bility and the anticipated high speciparticle with micrococcal nuclease ficity of cleavage of the hammerhead demonstrated a similar accessibility ribozyme makes it very attractive problem 29. Therefore, protein bound for targeted use. However, there are at the GUC cleavage site blocked other ribozyme catalytic sequences ribozyme activity while the CUC site which might prove amenable to was available for ribozyme cleavage. targeted use in rive in the near Work with antisense DNA oligo- future. Alterations in the original nucleotides has also shown that not Tetrahymena ribozyme might inall mRNA sites are equally available crease its specificity for in vivo apand effective as target sites 3°'31. So plications TM. A hairpin ribozyme far, the selection of ribozyme-access- has been described, with the active ible sites has been empirical. How- site derived from the minus strand of ever, we hope to find mRNA sites, the satellite of Tobacco Ringspot such as the translation initiation Virus 35'36. This ribozyme has a much codon, or the splicing and poly- higher catalytic activity than the adenylation sites, that are consistently hammerhead ribozyme in vitro 35, accessible to ribozyme cleavage. but so far has not been tested in the A second aspect of the ribozyme's presence of protein or within cells. inhibition activity that is less well There is self-cleaving RNA activity

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associated w i t h the hepatitis delta virus 37-39 and

self-cleaving d o m a i n is c o n t a i n e d on a transcript of n e w t satellite DNA 4°. Moreover, there is the well k n o w n cleavage activity associated w i t h RNase P (Ref. 2). It is possible that these cleavage d o m a i n s can be a d a p t e d for targeted r i b o z y m e s w i t h greater activity in eukaryotic cells t h a n has so far been s h o w n by the h a m m e r h e a d ribozyme. The h a m m e r h e a d r i b o z y m e c o u l d also be refined for m a m m a l i a n applications. So far we have o n l y considered the self-cleavage d o m a i n s that nature has supplied. These might well be i m p r o v e d u p o n by using a genetic selection protocol, w i t h cells p l a c e d u n d e r c o n d i t i o n s w h e r e high r i b o z y m e activity is r e q u i r e d for survival. It m a y also be possible to d e v e l o p in vitro selection cycles (evolution in a test tube?) to generate a better cleavage activity 41'42.

Acknowledgments

12 13 14 15

16 17

18 19 20 21

I w o u l d like to thank Max Birnstiel,

Ernst Wagner and the m e m b e r s of the Birnstiel group for their m a n y helpful suggestions. I appreciate the critical c o m m e n t s of A d r i a n Bird a n d Joan Boyes.

22

23

M. Cotten is at the Research Institute o f Molecular Pathology, Dr. Bohr-gasse 7, Vienna, Austria.

24

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25

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:: :

::i : i

:: i ::