Research in VeterinaryScience1991, 50, 131-133
Detection of foreign DNA in transgenic chicken embryos using the polymerase chain reaction D. SAVVA, N. PAGE, L. VICK, K. SIMKISS, School o f Animal and Microbial Sciences, University o f Reading, Whiteknights, PO Box 228, Reading RG6 2 A J
Chicken primordial germ cells were infected with a defective retrovirus containing the Escherichia coil iacZ gene and injected into the heart of stage 15 embryos. DNA samples were isolated from various tissues of the injected embryos at different stages of development and were examined for the presence of the lacZ gene using the polymerase chain reaction. Integration of the retrovirus DNA was demonstrated with a 32P-labelled oligonucleotide in five-, 10- and 18-day embryos. This quick procedure provides an opportunity for the early detection of foreign DNA in small numbers of transfected cells and is a valuable tool in the detection of transgenic animals.
THERE is currently a wide range of techniques being used to produce transgenic animals. They range from microinjection of DNA (deoxyribonucleic acid) into the pronuclei of the fertilised egg to infection of embryonic cells with defective retroviruses. One of the most promising of these methods uses pleuripotent stem ceils either from the inner cell mass of the mammalian embryo (Bradley et al 1984) or from the blastoderm of avian embryos (Petitte et al 1989). These cells can be injected with vector DNA, maintained in cell culture and then reintroduced into recipient embryos. If a clone of such cells can be maintained it opens up the possibility of producing offspring with chimaeric gonads derived from these stem cells. The authors have recently devised a variant of this procedure in which primordial germ cells from the embryo of the fowl are transfected with a defective retrovirus. These cells are then reintroduced into recipient embryos to produce a chick with a chimaeric gonad containing vector DNA (Simkiss et al 1990). One of the main problems in such studies is the detection of the foreign DNA. This is usually performed by Southern blotting (Southern 1975) and subsequent hybridisation with DNA probes but these techniques are time consuming and require relatively large amounts of DNA. This paper describes a method
which is applicable to a wide range of developmental studies since it enables the early and rapid screening of a small number of cells by the polymerase chain reaction (PER) (Saiki et al 1985). This opens up the possibility of tracing the fate of stem cell DNA during embryological development. Paradoxically, one of the main drawbacks of the PER reaction is its main attribute, that is, its extreme sensitivity. Consequently, in many instances contaminating host DNA is also amplified, mostly due to self-priming, while a number of false positive results can also be obtained for what are often quite bizarre reasons (Kitchin et al 1990). To overcome both of these problems sensitiv!ty of the PER and the specificity of hybridisation to a unique oligonucleotide have been combined. Thus the vector DNA introduced into primordial germ cells could be followed through to the formation of a normal gonad.
Materials and methods
Isolation and injection o f primordial germ cells Primordial germ cells (PGCs) were prepared by isolating the germinal crescent region of stage 12 embryos (approximately 48 hours incubation) and dissociating the tissue in calcium- and magnesiumfree saline containing 0-2 per cent w/v ethylene diamine tetra-acetic acid (EDTA). The cells were washed in Medium 199 with Earle's salts without Lglutamine (Sigma Chemical Company) and infected with a replication-defective form of the reticuloendothelial virus containing the lacZ gene (Meyers et al 1990) for 15 to 20 minutes. A suspension of the cells was stained with erythrosin B giving a viability count of approximately 90 per cent. Cells larger than 15 tzm diameter were considered to be PGCs and 50 to 100 of these were incubated in Dulbecco's modified Eagle's medium overnight or were injected with a glass capillary into a stage 15 embryo (50 to-55 hours incubation) by cardiac puncture.
131
132
D. Savva, N. Page, L. Vick, K. Simkiss 1
Preparation o f DNA Homogenised tissues or cell suspensions were centrifuged for five minutes in a microfuge and the cell pellet was washed in phosphate buffered saline. The washed cells were pelleted, resuspended in 40 #l sterile distilled water, boiled for 15 minutes and centrifuged to pellet the debris. The snpernatant was used as a source of crude DNA.
Amplification o f lacZ DNA using PeR B
The sequences of the two oligonucleotide primers (20mers) were based on the published sequence for the Escherichia eoli lacZ gene (Maizels 1973, Kalnins et al 1983) with one of these (DS44) being in the same sense as this sequence and the other (DS45) being of the opposite sense. Primer DS44 corresponds to six untranslated nucleotides and the first 14 translated c nucleotides of the lacZ gene (nucleotides 36 to 55 in the sequence of Maizels (1973); 5'GCTATGACCATGATTACGGA-3') and primer DS45 ( 5 ' - C A A A T T C A G A C G G C A A A C G A - 3 ' ) corresponds to nucleotides 514 to 495 in the sequence of Kalnins et al (1983). These oligonucleotides should therefore result in the amplification of a 519 base pair (bp) fragment of DNA. D PCR reactions were performed in a final volume of 100 #l as described by Savva et al (1990) except that the magnesium chloride concentration was 3 mM. Following the initial 'melting' of the DNA by incubating at 95°C for five minutes, 40 cycles of amplification were performed; each amplification cycle consisted of incubations at 95 ° C for one minute, 50°C for one minute and 74°C for two minutes. At E the end of the 40 cycles the reactions were incubated at 25°C for five minutes.
DNA
hybridisations
The probe used was an oligonucleotide (17mer) corresponding to nucleotides 54 to 38 of the lacZ gene (Kalnins et al 1983); its sequence was 5 ' GTTTTCCCAGTCACGAC-3'. This was radiolabeUed to a specific activity of 2.6 × 108 disintegrations per minute (dpm) using [.y_32p] ATP (3000 Ci mmole-1) and T4 polynucleotide kinases (both from Amersham Internati0nal). Amplified DNA was analysed for thepresence of l a e Z DNA using dot blots. The amplified DNA (25 /zl) was first denatured by incubation for 30 minutes at room temperature following the addition of 75 #1 20 x ssc (ssc is 150 mM sodium chloride, 15 mM trisodium citrate) and 50 #1 1 M sodium hydroxide. DNA samples were applied to a Hybond-N membrane (Amersham International).usinga Hybridot filtration
iiii!iiiili~i!ilii:i¸¸¸
FiG 1 : Dot blot hybridisation analysis of PCR products. DNA from various tissues was subjected to PCR to amplify a portion of the lacZ gene, dot-blotted on nylon membranes and hybridised to a 32p-labelled oligonucleotide specific for lacZ at 40°C in 2 × SSC containing O. 5 per cent (w/v) skimmed dried milk. (A) 1 and 2, uninfected 18-day embryos; 3 and 4, retrovirus vector. (B) 1 and 2, retrovirus-infected primordial germ cells; 3 and 4, uninfected primordial germ cells. (C) 1-4, 5 day infected transgenic embryos. (D) 1-4, 10 day transgenic embryos. (E) Muscle (1), liver (2), heart (3) and gonad (4} from an 18-day embryo
manifold (Gibco) and washed with 100 #1 of 20 x ssc. The membranewas baked at 80°C for one hour, irradiated on an ultraviolet transilluminator for 30 seconds and incubated in 2 × ssc containing 0' 5 per cent (w/v) skimmed dried milk at 40°C for two hours before the probe was added. Hybridisation was allowed to proceed overnight at 40°C and the membrane was washed twice (15 minutes each) in 2 × s s c . The
PCR to detect transgenic c h i c k e n s
washed m e m b r a n e was exposed to F u j i - R X X-ray film.
Results and discussion T h e PCR amplifies regions o f DNA between two oligonucleotide primers. In the experiments described here it was used to amplify a 519 b p f r a g m e n t of the E coil l a c Z gene which is carried on the defective retrovirus (Meyers et al 1990). The oligonucleotide p r o b e used for the hybridisations did not hybridise to the PCR p r o d u c t o b t a i n e d with chicken DNA c o n f i r m i n g b o t h its specificity a n d the absence Of vector DNA (Fig 1A). Since retroviruses possess an RNA g e n o m e they are n o t amplified until they are converted into the provirus a n d integrated into cellular DNA. The alkaline digestion in the h y b r i d i s a t i o n step ensures t h a t no RNA remains to hybridise to the oligonucleotide probe. T h u s , using a 32P-labelled region of the bacterial l a c Z gene to hybridise to samples o f nucleic acids produces n o specific binding with the defective vector. The oligonucleotide primers themselves give negative results with d o t blots (results not shown; however, all reactions in Fig 1 c o n t a i n e d b o t h DS44 a n d DS45). Once the defective retrovirus has been integrated into the g e n o m e o f a cell it i s capable o f being amplified by the PCR integration. T h u s p r i m o r d i a l germ cells t h a t have been isolated f r o m the stage 12 embryo, infected with the retroviral vector a n d m a i n tained in culture overnight to p e r m i t i n t e g r a t i o n give a positive assay (Fig 1B). These cells can subsequently be injected into a stage 15 e m b r y o a n d identified in the five- a n d 10-day e m b r y o (Fig 1C a n d D, respectively). As the o r g a n i s m grows a n d the cell lines b e c o m e progressively segregated into specific tissues the transfected DNA becomes restricted to the gonocytes o f the g o n a d s (Fig 1E)~ It is a p p a r e n t , therefore, t h a t a c o m b i n a t i o n o f the use o f retroviral vectors (Price 1987) a n d the PCR (Saiki et al 1985) provides a powerful way o f tracing cell lineages during development. The m a i n p r o b l e m with using these m e t h o d s is the possibility o f cross reactions which, with the powerful amplification involved in the PCR, can lead to false positive reactions. To o v e r c o m e this the h y b r i d i s a t i o n p r o b e was restricted to a short unique sequence in the l a c Z gene. U n d e r the h y b r i d i s a t i o n conditions used here, this oligonucleotide retained the ability to detect the l a c Z gene w i t h o u t any cross reaction with o t h e r DNA fractions.
133
In conclusion, procedures have been devised to follow the fate of foreign DNA in a few cells of chimaeric transgenic animals. By these m e a n s the a u t h o r s have been able to identify the retroviral provirus in infected p r i m o r d i a l germ cells, in early e m b r y o s a n d in the gonads o f 18-day chicken e m b r y o s t h a t were injected with transfected primordial germ cells.
Acknowledgements W e t h a n k G r a h a m Luke for technical assistance. This work was s u p p o r t e d by a g r a n t f r o m the Agricultural a n d F o o d Research Council (UK).
References BRADLEY, A., EVANS, M., KAUFMAN, M. H. & ROBERTSON, E. (1984) Formation of germ line chimeras from embryo derived teratocarcinoma cell lines. Nature 309, 255-256 KALNINS, A., OTTO, K., RUTHER, U. & MULLER-HILL, B. (1983) Sequence of the lacZ gene of Escherichia coli. EMBO Journal 2,593-597 KITCHIN, P. A., SZOTYORI, Z., FROMHOLC, C. &ALMOND, N. (1990)Avoidance of false positives. Nature 344, 201 MAIZELS, N. M. (1973) The nucleotide sequence of the lactose messenger ribonucleic acid transcribed from the UV5 promoter mutant of Escherichia coli. Proceedings of the National Academy of Scienee of the USA 70, 3585-3589 MEYERS, N., BUMSTEAD, N. & BOOTH, S. (1990) Development and testing of an improved helper cell line for avian reticuloendotheliosis virus-based vectors. 4th World Congress Genetics Applied Livestock Production 16, 131-134 PETITTE, J. N., CLARK, M. E., LIU, G., VERRINDER GIBBINS, A. M. & ETCHES, R. J. (1989) Production of somatic and germline chimeras in the chicken by transfer of early blastodermal cells. Development 108, 185-189 PRICE, J. (1987) Retrovirusesand the study of cell lineage. Development 101,409-419 SAIKI, R. K., SCHARF, S., FALOONA, F., MULLIS, K. B., HORN, G. T. & ERLICH, H. A. (1985) Enzymatic amplification of ~3-globingenomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230, 1350-1354 SAVVA, D., TARNOKY, A. L. & VICKERS, M. F. (1990) Genetic characterisation of an alloalbumin, albumin Kashmir, using gene amplification and allele-specific oligonucleotides. Biochemical Journal 266, 615-617 SIMKISS, K., VICK, L., LUKE, G., PAGE, N. & SAVVA, D. (1990) Infection of primordial germ cells with defective retrovirus and their transfer to the developing embryo. 4th World Congress Genetics Applied Livestock Production 16, 111-114 SOUTHERN, E. M. (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. Journal of Molecular Biology 98,503- 517
Received May 21, 1990 Accepted June 20, 1990
Detection of foreign DNA in transgenic chicken embryos using the polymerase chain reaction D. SAVVA, N. PAGE, L. VICK, K. SIMKISS, School o f Animal and Microbial Sciences, University o f Reading, Whiteknights, PO Box 228, Reading RG6 2 A J
Chicken primordial germ cells were infected with a defective retrovirus containing the Escherichia coil iacZ gene and injected into the heart of stage 15 embryos. DNA samples were isolated from various tissues of the injected embryos at different stages of development and were examined for the presence of the lacZ gene using the polymerase chain reaction. Integration of the retrovirus DNA was demonstrated with a 32P-labelled oligonucleotide in five-, 10- and 18-day embryos. This quick procedure provides an opportunity for the early detection of foreign DNA in small numbers of transfected cells and is a valuable tool in the detection of transgenic animals.
THERE is currently a wide range of techniques being used to produce transgenic animals. They range from microinjection of DNA (deoxyribonucleic acid) into the pronuclei of the fertilised egg to infection of embryonic cells with defective retroviruses. One of the most promising of these methods uses pleuripotent stem ceils either from the inner cell mass of the mammalian embryo (Bradley et al 1984) or from the blastoderm of avian embryos (Petitte et al 1989). These cells can be injected with vector DNA, maintained in cell culture and then reintroduced into recipient embryos. If a clone of such cells can be maintained it opens up the possibility of producing offspring with chimaeric gonads derived from these stem cells. The authors have recently devised a variant of this procedure in which primordial germ cells from the embryo of the fowl are transfected with a defective retrovirus. These cells are then reintroduced into recipient embryos to produce a chick with a chimaeric gonad containing vector DNA (Simkiss et al 1990). One of the main problems in such studies is the detection of the foreign DNA. This is usually performed by Southern blotting (Southern 1975) and subsequent hybridisation with DNA probes but these techniques are time consuming and require relatively large amounts of DNA. This paper describes a method
which is applicable to a wide range of developmental studies since it enables the early and rapid screening of a small number of cells by the polymerase chain reaction (PER) (Saiki et al 1985). This opens up the possibility of tracing the fate of stem cell DNA during embryological development. Paradoxically, one of the main drawbacks of the PER reaction is its main attribute, that is, its extreme sensitivity. Consequently, in many instances contaminating host DNA is also amplified, mostly due to self-priming, while a number of false positive results can also be obtained for what are often quite bizarre reasons (Kitchin et al 1990). To overcome both of these problems sensitiv!ty of the PER and the specificity of hybridisation to a unique oligonucleotide have been combined. Thus the vector DNA introduced into primordial germ cells could be followed through to the formation of a normal gonad.
Materials and methods
Isolation and injection o f primordial germ cells Primordial germ cells (PGCs) were prepared by isolating the germinal crescent region of stage 12 embryos (approximately 48 hours incubation) and dissociating the tissue in calcium- and magnesiumfree saline containing 0-2 per cent w/v ethylene diamine tetra-acetic acid (EDTA). The cells were washed in Medium 199 with Earle's salts without Lglutamine (Sigma Chemical Company) and infected with a replication-defective form of the reticuloendothelial virus containing the lacZ gene (Meyers et al 1990) for 15 to 20 minutes. A suspension of the cells was stained with erythrosin B giving a viability count of approximately 90 per cent. Cells larger than 15 tzm diameter were considered to be PGCs and 50 to 100 of these were incubated in Dulbecco's modified Eagle's medium overnight or were injected with a glass capillary into a stage 15 embryo (50 to-55 hours incubation) by cardiac puncture.
131
132
D. Savva, N. Page, L. Vick, K. Simkiss 1
Preparation o f DNA Homogenised tissues or cell suspensions were centrifuged for five minutes in a microfuge and the cell pellet was washed in phosphate buffered saline. The washed cells were pelleted, resuspended in 40 #l sterile distilled water, boiled for 15 minutes and centrifuged to pellet the debris. The snpernatant was used as a source of crude DNA.
Amplification o f lacZ DNA using PeR B
The sequences of the two oligonucleotide primers (20mers) were based on the published sequence for the Escherichia eoli lacZ gene (Maizels 1973, Kalnins et al 1983) with one of these (DS44) being in the same sense as this sequence and the other (DS45) being of the opposite sense. Primer DS44 corresponds to six untranslated nucleotides and the first 14 translated c nucleotides of the lacZ gene (nucleotides 36 to 55 in the sequence of Maizels (1973); 5'GCTATGACCATGATTACGGA-3') and primer DS45 ( 5 ' - C A A A T T C A G A C G G C A A A C G A - 3 ' ) corresponds to nucleotides 514 to 495 in the sequence of Kalnins et al (1983). These oligonucleotides should therefore result in the amplification of a 519 base pair (bp) fragment of DNA. D PCR reactions were performed in a final volume of 100 #l as described by Savva et al (1990) except that the magnesium chloride concentration was 3 mM. Following the initial 'melting' of the DNA by incubating at 95°C for five minutes, 40 cycles of amplification were performed; each amplification cycle consisted of incubations at 95 ° C for one minute, 50°C for one minute and 74°C for two minutes. At E the end of the 40 cycles the reactions were incubated at 25°C for five minutes.
DNA
hybridisations
The probe used was an oligonucleotide (17mer) corresponding to nucleotides 54 to 38 of the lacZ gene (Kalnins et al 1983); its sequence was 5 ' GTTTTCCCAGTCACGAC-3'. This was radiolabeUed to a specific activity of 2.6 × 108 disintegrations per minute (dpm) using [.y_32p] ATP (3000 Ci mmole-1) and T4 polynucleotide kinases (both from Amersham Internati0nal). Amplified DNA was analysed for thepresence of l a e Z DNA using dot blots. The amplified DNA (25 /zl) was first denatured by incubation for 30 minutes at room temperature following the addition of 75 #1 20 x ssc (ssc is 150 mM sodium chloride, 15 mM trisodium citrate) and 50 #1 1 M sodium hydroxide. DNA samples were applied to a Hybond-N membrane (Amersham International).usinga Hybridot filtration
iiii!iiiili~i!ilii:i¸¸¸
FiG 1 : Dot blot hybridisation analysis of PCR products. DNA from various tissues was subjected to PCR to amplify a portion of the lacZ gene, dot-blotted on nylon membranes and hybridised to a 32p-labelled oligonucleotide specific for lacZ at 40°C in 2 × SSC containing O. 5 per cent (w/v) skimmed dried milk. (A) 1 and 2, uninfected 18-day embryos; 3 and 4, retrovirus vector. (B) 1 and 2, retrovirus-infected primordial germ cells; 3 and 4, uninfected primordial germ cells. (C) 1-4, 5 day infected transgenic embryos. (D) 1-4, 10 day transgenic embryos. (E) Muscle (1), liver (2), heart (3) and gonad (4} from an 18-day embryo
manifold (Gibco) and washed with 100 #1 of 20 x ssc. The membranewas baked at 80°C for one hour, irradiated on an ultraviolet transilluminator for 30 seconds and incubated in 2 × ssc containing 0' 5 per cent (w/v) skimmed dried milk at 40°C for two hours before the probe was added. Hybridisation was allowed to proceed overnight at 40°C and the membrane was washed twice (15 minutes each) in 2 × s s c . The
PCR to detect transgenic c h i c k e n s
washed m e m b r a n e was exposed to F u j i - R X X-ray film.
Results and discussion T h e PCR amplifies regions o f DNA between two oligonucleotide primers. In the experiments described here it was used to amplify a 519 b p f r a g m e n t of the E coil l a c Z gene which is carried on the defective retrovirus (Meyers et al 1990). The oligonucleotide p r o b e used for the hybridisations did not hybridise to the PCR p r o d u c t o b t a i n e d with chicken DNA c o n f i r m i n g b o t h its specificity a n d the absence Of vector DNA (Fig 1A). Since retroviruses possess an RNA g e n o m e they are n o t amplified until they are converted into the provirus a n d integrated into cellular DNA. The alkaline digestion in the h y b r i d i s a t i o n step ensures t h a t no RNA remains to hybridise to the oligonucleotide probe. T h u s , using a 32P-labelled region of the bacterial l a c Z gene to hybridise to samples o f nucleic acids produces n o specific binding with the defective vector. The oligonucleotide primers themselves give negative results with d o t blots (results not shown; however, all reactions in Fig 1 c o n t a i n e d b o t h DS44 a n d DS45). Once the defective retrovirus has been integrated into the g e n o m e o f a cell it i s capable o f being amplified by the PCR integration. T h u s p r i m o r d i a l germ cells t h a t have been isolated f r o m the stage 12 embryo, infected with the retroviral vector a n d m a i n tained in culture overnight to p e r m i t i n t e g r a t i o n give a positive assay (Fig 1B). These cells can subsequently be injected into a stage 15 e m b r y o a n d identified in the five- a n d 10-day e m b r y o (Fig 1C a n d D, respectively). As the o r g a n i s m grows a n d the cell lines b e c o m e progressively segregated into specific tissues the transfected DNA becomes restricted to the gonocytes o f the g o n a d s (Fig 1E)~ It is a p p a r e n t , therefore, t h a t a c o m b i n a t i o n o f the use o f retroviral vectors (Price 1987) a n d the PCR (Saiki et al 1985) provides a powerful way o f tracing cell lineages during development. The m a i n p r o b l e m with using these m e t h o d s is the possibility o f cross reactions which, with the powerful amplification involved in the PCR, can lead to false positive reactions. To o v e r c o m e this the h y b r i d i s a t i o n p r o b e was restricted to a short unique sequence in the l a c Z gene. U n d e r the h y b r i d i s a t i o n conditions used here, this oligonucleotide retained the ability to detect the l a c Z gene w i t h o u t any cross reaction with o t h e r DNA fractions.
133
In conclusion, procedures have been devised to follow the fate of foreign DNA in a few cells of chimaeric transgenic animals. By these m e a n s the a u t h o r s have been able to identify the retroviral provirus in infected p r i m o r d i a l germ cells, in early e m b r y o s a n d in the gonads o f 18-day chicken e m b r y o s t h a t were injected with transfected primordial germ cells.
Acknowledgements W e t h a n k G r a h a m Luke for technical assistance. This work was s u p p o r t e d by a g r a n t f r o m the Agricultural a n d F o o d Research Council (UK).
References BRADLEY, A., EVANS, M., KAUFMAN, M. H. & ROBERTSON, E. (1984) Formation of germ line chimeras from embryo derived teratocarcinoma cell lines. Nature 309, 255-256 KALNINS, A., OTTO, K., RUTHER, U. & MULLER-HILL, B. (1983) Sequence of the lacZ gene of Escherichia coli. EMBO Journal 2,593-597 KITCHIN, P. A., SZOTYORI, Z., FROMHOLC, C. &ALMOND, N. (1990)Avoidance of false positives. Nature 344, 201 MAIZELS, N. M. (1973) The nucleotide sequence of the lactose messenger ribonucleic acid transcribed from the UV5 promoter mutant of Escherichia coli. Proceedings of the National Academy of Scienee of the USA 70, 3585-3589 MEYERS, N., BUMSTEAD, N. & BOOTH, S. (1990) Development and testing of an improved helper cell line for avian reticuloendotheliosis virus-based vectors. 4th World Congress Genetics Applied Livestock Production 16, 131-134 PETITTE, J. N., CLARK, M. E., LIU, G., VERRINDER GIBBINS, A. M. & ETCHES, R. J. (1989) Production of somatic and germline chimeras in the chicken by transfer of early blastodermal cells. Development 108, 185-189 PRICE, J. (1987) Retrovirusesand the study of cell lineage. Development 101,409-419 SAIKI, R. K., SCHARF, S., FALOONA, F., MULLIS, K. B., HORN, G. T. & ERLICH, H. A. (1985) Enzymatic amplification of ~3-globingenomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230, 1350-1354 SAVVA, D., TARNOKY, A. L. & VICKERS, M. F. (1990) Genetic characterisation of an alloalbumin, albumin Kashmir, using gene amplification and allele-specific oligonucleotides. Biochemical Journal 266, 615-617 SIMKISS, K., VICK, L., LUKE, G., PAGE, N. & SAVVA, D. (1990) Infection of primordial germ cells with defective retrovirus and their transfer to the developing embryo. 4th World Congress Genetics Applied Livestock Production 16, 111-114 SOUTHERN, E. M. (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. Journal of Molecular Biology 98,503- 517
Received May 21, 1990 Accepted June 20, 1990
