Proc. Natl. Acad. Sci. USA Vol. 75, No. 1, pp. 495-499, January 1978
Microbiology
Characterization and mapping o RNaseTi-resistant oligonucleotides derived from the genomes of Akv and MCF murine leukemia viruses (recombination)
JEAN ROMMELAERE, DOUGLAS V. FALLER, AND NANCY HOPKINS* Department of Biology and Center for Cancer Research, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139
Communicated by Wallace P. Rowe, October 6, 1977
Ti RNA fingerprints of the genomes of Akv-i ABSTRACT and Akv-2 C-type viruses are indistinguishable and oligonucleotide maps of these viruses are probably the same. Akv-i and -2 share 55-75% of their large Ti-resistant oligonucleotides with four MCF viruses isolated from AKR mice or from NIH Swiss mice that inherit either the Akv-1 or Akv-2 virus-inducing locus of AKR. The majority of Akv oligonucleotides missing from Ti fingerprints of MCFs and the majority of oligonucleotides unique to MCF viruses are clustered and lie at corresponding positions in the 3' half of the oligonucleotide maps of Akv and MCF viruses. The RNA sequences present in different MCF isolates but not present in Akv viruses are related. These results are consistent with a recombinational origin of MCF viruses, as proposed by Hartley and Rowe and their collaborators.
Akv-1 or Akv-2 (1, 2) and MCF (3) viruses can be isolated from mice of the high leukemic AKR strain. Contrary to Akv-1 or -2 viruses, which are present in the mice irrespective of age or tissue and are N-tropic ecotropic viruses (4), MCF viruses are isolated from lymphoid tissue of late preleukemic or leukemic mice, have a dual (exo/xenotropic) host range and other envelope-determined properties, and induce foci in cultured mink cells. To explain the dual host range of MCF viruses and to explain their isolation both from AKR mice and from NIH Swiss mice that inherit either the Akv-1 or Akv-2 virus-inducing locus of AKR, Hartley et al. (3) proposed that MCF viruses may arise in vivo by recombination between an Akv-i or -2 virus and an as yet unidentified xenotropic virus. To investigate this proposal we used Ti RNA fingerprinting and oligonucleotide mapping to analyze the genomes of Akv-i or -2 viruses and of four MCF viruses. Two of the MCF isolates, MCF 13 and MCF 247, were obtained from AKR mice, and two, MCF Vl-36 and MCF V2-34, were obtained from NIH Swiss mice inheriting the Akv-1 or Akv-2 locus of AKR, respectively. Since we have already reported that the large Ti-resistant oligonucleotides of Akv-I and -2 viruses are indistinguishable (5), these viruses will be referred to indiscriminately as Akv viruses where appropriate. MATERIALS AND METHODS Cells and Viruses. The origin of Akv-I and Akv-2 viruses has been described (1, 2, 5). MCF viruses were obtained from old preleukemic AKR mice (MCF 13 and MCF 247) or leukemic. NIH Swiss mice inheriting the Akv-1 (MCF V1-36) or Akv-2 (MCF V2-34) virus-inducing locus of AKR and were purified by successive end-point dilutions. Akv and MCF viruses were kindly provided by J. W. Hartley and W. P. Rowe. SC-i cells (6) chronically infected with each virus were used to prepare 32P-labeled viral RNA. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertsement" in accordance with 18 U. S. C. §1734 solely to indicate
this fact.
Ti RNA Fingerprinting and Oligonucleotide Mapping. Procedures for preparation of 32P-labeled viral RNA, Ti RNA fingerprinting, quantitative and qualitative visual analyses of the products of pancreatic RNase digestion of Ti oligonucleotides (7), and oligonucleotide mapping (refs. 8 and 12; Faller and Hopkins, unpublished data) have been described in detail. The following limitations of the methods of RNA fingerprinting and oligonucleotide mapping should be recalled: (i) Only approximately 5% of the viral genome is contained in the analyzed Ti-resistant oligonucleotides (8); thus critical regions of the genomes may go undetected. (ii) The position of an oligonucleotide on the oligonucleotide maps should in general only be considered accurate to ±1-2 oligonucleotides towards the 3' end of the genome and +2-3 towards the 5' end. (iii) An oligonucleotide map reveals relative, not absolute, positions between Ti oligonucleotides and should only be considered an approximate physical map of the viral genome (see ref. 8).
RESULTS Akv Viruses Share Approximately 55-75% of Their Large RNase Ti-Resistant Oligonucleotides with MCF 13, MCF 247, MCF Vi-36, and MCF V2-34. SC-I cells chronically infected with Akv-i, Akv-2, MCF 13, MCF 247, MCF V1-36, and MCF V2-34 viruses were used to prepare uniformly 32P-labeled 70S viral RNA. The RNA was digested with RNase T1, and the
resulting oligonucleotides were separated by two-dimensional gel electrophoresis. Autoradiograms of the second dimension gels, termed T1 fingerprints, are shown in Fig. 1 A and B for Akv-2 and MCF 247 viruses and diagrams of these fingerprints are shown in Fig. 1 C and D. Diagrams of the T1 fingerprints of MCF 13, MCF VI-36, and MCF V2-34 are shown in Fig. 2.
That the arbitrarily numbered spots in Figs. 1 C and D and 2 represent oligonucleotides derived from the viral genome is inferred from the facts that T1 fingerprints of 70S viral RNA were indistinguishable from those of 35S RNA (data not shown) and that the numbered oligonucleotides are (with two exceptions in MCF VI-36, see legend to Fig. 2) present in molar amount relative to one another in the fingerprints. Molarity of oligonucleotides was determined by quantitation of the radioactivity present in each oligonucleotide in the fingerprints (data not shown) combined with a determination of their length as described below. Since oligonucleotides that differ in sequence or base composition can possess similar electrophoretic mobilities in this system, to ascertain which oligonucleotides of Akv viruses are also present in T1 fingerprints of MCF viruses we further analyzed oligonucleotides of Akv and MCF viruses that appear to have the same electrophoretic mobility. Ti oligonucleotides *
495
To whom reprint requests should be addressed.
496
Proc. Natl. Acad. Sci. USA 75 (1978)
Microbiology: Rommelaere et al.
* * 0
-I'
0 0
000 64?
Ak
A
0
Akv-2
MCF 247
B
0
00~~
2
Akv-2
MCF
247
----------------I
FIG. 1. Two-dimensional gel electrophoretic T1 fingerprints of 70S RNA of Akv-2 and MCF 247 viruses. T1 fingerprints of (A) Akv-2 70S [32P]RNA (2.8 X 106 cpm) and (B) MCF 247 70S [32P]RNA (1.7 X 106 cpm). Autoradiographs were exposed for 26 (Akv-2) and 44 (MCF) hr. (C and D) Diagrams of A and B, respectively. Spot numbers are arbitrary. Open circles in C represent oligonucleotides that are present in molar amount relative to one another. Open circles in D represent single oligonucleotides that are common to Akv and MCF. Black circles (D) represent single oligonucleotides that are found in the fingerprint of MCF but not Akv. Circles that are half white and half black represent spots that consist of two oligonucleotides that comigrate, one of which is probably present in Akv and one of which is MCF-specific. Areas or spots that do not contain a single molar oligonucleotide are cross-hatched. XC and B indicate positions of dye markers xylene cyanol FF and bromphenol blue, respectively. Arrows indicate direction of migration of first and second electrophoresis.
removed from the second dimension gels and digested with pancreatic RNase and the products were separated as described (6). In addition to providing identification of each oligonucleotide, quantitation of these secondary digestion products allowed a determination of their length (h15%) and, in conjunction with quantitation of the radioactivity present in each oligonucleotides in each fingerprint, allowed a determination of their relative molarities. The results of the quantitative analysis of the products of pancreatic RNase digestion of T1 oligonucleotides of Akv and MCF viruses are shown in Table 1 (and ref. 5). These data (or in some cases, qualitative analysis of the secondary digestion products) provide the justification for the diagrams in Figs. 1 C and D and 2. In these diagrams, T1 oligonucleotides that are common to Akv viruses and to a particular MCF virus are represented by open circles. Oligonucleotides that are found in MCF viruses but not in Akv viruses are represented by black circles and are numbered 101, 102, etc. The latter will be referred to as MCF-specific or Moligonucleotides. Of the 41 large Tl-resistant oligonucleotides of Akv viruses that were analyzed, MCF 13 possesses 22/41 or 54%; MCF 247, 30/41 or 74%; MCF V1-36, 29/41 or 70%; and MCF V2-34, 23/41 or 56%. Eight Akv oligonucleotides are not present in any of the four MCF isolates. Of the 25 analyzable M-oligonucleotides in the four MCF viruses (Table 2), one, possibly two, are shared by all four MCF isolates; two, possibly three, are were
shared by three of the four isolates; and six are shared by two of the four viruses. Oligonucleotide Maps of Akv-1, Akv-2, MCF 247, and MCF V2-34. If MCF viruses arise by recombination, then one might expect that M-oligonucleotides and also the Akv oligonucleotides that are missing in T1 fingerprints of each MCF virus would be derived from one or just a few (corresponding) regions of the viral genomes. In particular, since MCF viruses possess altered gp7O proteins relative to Akv viruses (9), and since genetic evidence indicates that gp7O coding sequences of avian (10, 11) and murine (Faller and Hopkins, unpublished data) C-type viruses lie towards the 3' end of the viral RNA, one might expect that M-oligonucleotides would be derived from this region of the MCF genomes. To investigate this possibility we determined the physical order of the T1 oligonucleotides of Akv-i, Akv-2, MCF 247, and MCF V2-34 viruses by determining their relative distances from the 3' ends of the viral RNAs. The methodology used to construct the oligonucleotide maps was similar to that established by Coffin and Billeter (8) and by Wang et al. (12) and identical to that of Faller and Hopkins (unpublished data). The method involves determination of the order in which oligonucleotides are lost from T1 fingerprints of increasingly smaller size classes of poly(A)containing viral RNA. The oligonucleotide maps of Akv-i, MCF 247, and MCF V2-34 are shown in Fig. 3A. [A "crude" map of Akv-2 was obtained from a single experiment (data not
Microbiology: Rommelaere et al.
Proc. Natl. Acad. Sci. USA 75 (1978)
497
-Table 1. Products of pancreatic RNase digestion of large T1 oligonucleotides of Akv and MCF viruses
Oligonucleotide no.* (A)
36 37 38 38a 39 42
(B)
101 102 103 104 105 106 107 110
44
111 112 113 114
(C) (D)
(E)
FIG. 2. Schematic representation of T1 fingerprints of (A) MCF 13 70S [32P]RNA, (B) MCF V1-36, and (C) MCF V2-34. Symbols as in Fig. 1D. [Note that oligonucleotides 1 and 30 of MCF V1-36 are present in less than molar amount, implying that this virus is a mixture of two viruses differing at their 5' end (see Fig. 3A).]
shown) and suggested that corresponding Ti oligonucleotides of Akv-1 and Akv-2 occupy the same relative positions on the genomes of these viruses. ] Not all the Akv or M-oligonucleotides that were analyzed (Table 1 and ref. 5) could be mapped, since, in general, only the order of oligonucleotides that are well separated in a fingerprint can be determined accurately. In some cases, it became possible to map an Akv oligonucleotide that did not possess a unique
115 116 119 120 37 + 37a 38 + 38a 39 + 39a 44+47 3 + 109 9+ 108 12 + 122 35 + 121 107 + 107a 123 + 123a
Composition
5U,1OC,(AU),(A2C),(A2U),(A2G) 2U,7C,3(AC),(AU),G 5U,7C,(AC),(AU),(A2U),G 8U,7C,3(AC),(AG) U,5C,3(AC),(AU),(A3G) 2U,7C,3(AC),(AG) 4U,2C(A2C),(A3C),(A4C) 3U,7C,(AC),(AU),(A2C),(A2U),(A3U),(AG) 4U,1OC,(AC),2(A2C),G 11U,7C,3(AC),(AG) 7U,6C,(AC),(A2U),(A2G) 5U,8C,(AC),G 2U,5C,3(AC),(A4C),G 5U,C,(AU),(A4U),G 2U,10C,(AC),(A2C),G 4U,9C,(AC),(A2C),(AG) 5U,7C,(AC),(AU),(A2U),(A4G) 7U,6C,2(AC),2(AU),(A2C),G 9U,3C,2(AC),2(AU),2(A2U),G
U,3C,(AC),(AU),(A2C),(A3U),(A4C),(A4G) 2U,9C,2(AC),(A2C),G 1OU,5C,2(AC),(AU),(A2U),G 4U,4C,(AC),(AU),(A2C),G 5U,13C,4(AC),(AU),2(A2C),(A2U),G(AG) 11U,13C,3(AC),2(AU),(A2U),G,(AG) 2U,8C,5(AC),(AU),(A2U),(A3G),(A4G) 6U,7C,2(AC),4(AU),(A2C),(A3C),G,(A4G) 6U,16C,2(AC),2(AU),2(A4C),G,(A2G) 13U,11C,3(AC),2(AU),2(A2C),(A2U),(A3C), G,(AG) 6U,23C,(AC),3(AU),(A3C),(AG),G 6U,12C,2(AC),(AU),2(A2C),(AG),G
9U,7C,2(AU),(A4U),2G 5U,7C,2(AC),7(AU),(A3G),G
Average values of at least two determinations, normalized to the yield of the G-containing digestion product and rounded to the nearest integer are given. Approximately 200 cpm per nucleotide were typically found (from 20 X 106 cpm of 70S viral RNA). When the products of pancreatic RNase digestion of homologous oligonucleotides of Akv and/or MCF viruses were indistinguishable (spots that have the same number in the diagrams in Figs. 1 and 2), they are listed only once, or not at all if the products of the Akv oligonucleotide were reported previously (5). (A) Oligonucleotides with unique electrophoretic mobilities in fingerprints of (an) MCF but not an Akv virus (digestion products contained among products of corresponding Akv doublet or complex region); (B) M-oligonucleotides with unique electrophoretic mobilities; (C) oligonucleotides that do not possess unique electrophoretic mobilities in fingerprints of Akv (or MCF); (D) oligonucleotides of MCF that do not possess unique electrophoretic mobilities, one component also present in Akv; and (E) M-oligonucleotides that do not possess unique electrophoretic mobilities. * See diagrams, Figs. 1 and 2.
electrophoretic mobility in fingerprints of Akv viruses because the contaminating Akv oligonucleotide(s) was missing in the fingerprint of one of the two MCF viruses whose T1 oligonucleotides were ordered. The three oligonucleotide maps shown (Fig. 3A) are consistent with the assumption that oligonucleotides that are shared by Akv and MCF viruses occupy the same relative positions on the genomes of these viruses. Thus, to oh. tain a more complete Akv map, we took the libery of pooling the data pertaining to Akv oligonucleotides that were obtained from all the mapping experiments and constructed an oligonucleotide map of Akv referred to as the "pooled data" map
498
Proc. Natl. Acad. Sci. USA 75 (1978)
Microbiology: Rommelaere et al.
Table 2. Distribution of MCF-specific T1 oligonucleotides among four MCF viruses M-Oligonucleotide Cq 0
M
o
+ +
+
+
4
MCF MCF MCF MCF
13 247 V1-36 V2-34
+
l 0
0
+ +
+
+
+
+
LO
?* +
t-
to 0
0
0
+ +
+ +
+
,4 00 C1 0 M M 0 0 vo o '-4 "+ +
u LO
Nil
,
O
,-4
r
4
+
+
+
? ?
+ +
+
0 Cq
H. Cq
Cq
Cq
C Cq
et Cq
Cq
+ +
+
+
+
+
?
e
C1
+
+ +
+
+
+
+
List of MCF-specific oligonucleotides analyzed in all four MCF isolates; their presence in each isolate is indicated by a +. * An oligonucleotide is present at the corresponding electrophoretic position but does not possess a unique electrophoretic mobility and its products of pancreatic RNase digestion are insufficiently distinctive compared to those of the contaminating oligonucleotide(s) to allow unequivocal identification.
(Fig. 3 B and C). Using this map (Fig. 3B) we then identified Akv oligonucleotides that are missing in T1 fingerprints of each MCF virus. These oligonucleotides are indicated by black circles in Fig. 3B. It is apparent that the majority of Akv oligonucleotides that are missing in each MCF virus are clustered on the Akv genome and that the region between map positions 21 and 27 is missing in all four MCF viruses. In Fig. 3C we have indicated the relative positions (on the Akv pooled data map) of those M-oligonucleotides of MCF 247 and MCF V2-34 that could be mapped (black triangles). It is apparent from Fig. 3C (and Fig. 3A) that the majority of these M-oligonucleotides are clustered and lie in the regions of the maps corresponding to those "vacated" by the Akv oligonucleotides.
DISCUSSION We used T1 RNA fingerprinting and oligonucleotide mapping to investigate genetic relationship between Akv and MCF viruses. Our data indicate (i) that the four MCF viruses studied share between 55 and 75% of their T1 oligonucleotides with Akv viruses; (ii) that each MCF isolate is unique; (iii) that the Akv oligonucleotides missing in MCF viruses lie in the 3' half of the Akv oligonucleotide map; and (iv) that the MCF-specific RNA
A
Akv MCF 247 MCF V2-34
sequences present in the four MCF isolates are related and, in the two MCF viruses whose oligonucleotide maps were constructed, these sequences lie at positions on the MCF genomes corresponding to the regions of the missing Akv T1 oligonucleotides. That four MCF isolates differ would seem to support the suggestion that MCFs are not integrated in the mouse genome but arise by recombination involving an Akv virus (3). It is interesting to consider the data further in terms of this proposal. Clusters of M-Oligonucleotides and of Akv Oligonucleotides Missing in MCFs. The majority of M-oligonucleotides present in and the majority of Akv oligonucleotides missing from each MCF isolate are clustered on the oligonucleotide maps of MCF 247, MCF V2-34, and Akv (Fig. 3). (Note, however, that we cannot be certain that these clusters are uninterrupted.) Several explanations can be invoked to explain the presence of black spots and triangles (Fig. 3 B and C) outside these clusters: (i) MCF 13, MCF 247, and MCF V2-34 result from multiple cross-overs between Akv and a virus(es) that shares few or no Ti oligonucleotides with Akv. (ii) The nonAkv, putative xenotropic, parents of MCF viruses share some Ti oligonucleotides with Akv viruses and one or two cross-over events can result in the patterns observed in Fig. 3 B and C. (iii) The inaccuracies inherent in the method of oligonucleotide
5' (1-30) *21- (2-12-15) *27-16-29-5-7-24-4-6-23-28-20-17-25- (11-10) *3- (9-8) -2235- (19-18) *33-26-14 (1-21-30)*2-2- (27-15) -fi1-29 (^65)*7- (438- (37)*6) 4*-1-1 -B08-2- (11-1-19)*33-26-1 (30-1) *1-2- (21-15-V) -39-42-29-4-16-5-1-7-24-4-3 (1- 102-M) *13-15-14-11-26-14
3'
Akv OLIGO. 30( 1-21 21i2)15Q27-39-42(16-4429)(^65) 7-24(37-38- 4- 6)23-282-17-25(u-10) 3- 9- 8-2-35(19 18)33-26-14 1 2 3 4 5 6 7 8 910 2 141516 819 122 231233343537 POSITION
MCF 13
___
B MCF 247
000-000
0
--* ----
MCF V1-36
*--
MCF V2-34
*-..00----..09
MCF 247
v
V
V
0
V
. 0V
V
C MCF V2-34
V
V
V
V
V
V V vV -
FIG. 3. Oligonucleotide maps of Akv-1, MCF 247, and MCF V2-34. (A) Oligonucleotide maps constructed from relative yields of T1 oligonucleotides in fingerprints of fragmented poly(A)-containing RNA of different size classes. Maps of Akv-1, MCF 247, and MCF V2-34 are based on data from, respectively, two, two, and one experiment; eight, seven, and four size classes of RNA were used. Discrepancies between experiments preclude determining the relative order of oligonucleotides within parentheses. MCF-specific oligonucleotides are underlined. Distance between oligonucleotides is arbitrary. (B) "Pooled data" oligonucleotide map (see text) of Akv and diagrams indicating oligonucleotides missing in MCF viruses. The relative positions of 37 Akv T1 oligonucleotides were obtained as described in the text. Absence of an Akv oligonucleotide in T1 fingerprints of MCF viruses is indicated by a black dot; presence of Akv oligonucleotides in MCF viruses is indicated by a line. (C) Relative positions of MCF-specific oligonucleotides (v) on Akv pooled data map. Obtained by comparison of A and B. Spacing between triangles is arbitrary.
Microbiology:
Rommelaere et al.
mapping might explain some of the gaps in Fig. 3 B an uC(rbi example, positions 20, 29, and 36 in MCF 13; see Fig. SB). However, it is unlikely that mapping errors explain the positions outside clusters of the M-oligonucleotides or missing Akv oligonucleotides in MCF 247 or MCF V2-34 (Fig. 3 B and C). (iv) Since single base changes can cause pligonucleotide spots to appear or disappear in a fingerprint (7), it is possible that mutations could explain the location of some of the isolated black spots and triangles in Fig. 3 B and C. However, in those cases where M-oligonucleotides map at positions corresponding to missing isolated Akv oligonucleotides, where the sequences of the M-oligonucleotides are unrelated to those of the "replaced" Akv oligonucleotides (as revealed by Table 1, or ref. 5) and where the M-oligonucleotide appears in more than one MCF virus, we consider this explanation unlikely (for example, positions 32 and 37 in MCF 247). It is interesting to note that the M-oligonucleotides at map positions 32 and 37 of MCF 247 are not alleles of the Akv oligonucleotides missing from the corresponding positions: MCF 13 possesses both the Akv and the M-oligonucleotides that map at position 32 (Table 2); endogenous ecotropic viruses of BALB/c possess both the Akv and the M-oligonucleotides located at position 37 (13). Assuming that the non-Akv parents of MCFs do not possess the Akv oligonucleotide at position 20, the data in Fig. 3B reveal a preferred region, between positions 20 and 21, for one of the proposed recombination events generating MCF viruses. However, because of the limitations of the methods of analysis, we cannot conclude that this point is identical in three MCFs (of four, if one includes MCF 13 by assuming correct placement of the Akv oligonucleotide at position 20; see Fig. 3B). The Putative Xenotropic Parents of Different MCF Isolates Are Related. Our data reveal that the non-Akv RNA sequences present in the genomes of different MCF isolates, whether derived from AKR mice or from NIH congenics, are related. The data do not allow us to conclude whether or not these sequences are identical. Of the four clustered M-oligonucleotides represented by black triangles in the oligonucleotide map of MCF 247 (Fig. 3C), at least two, probably three, are also present in MCF V2-34; the most 5' of the four is not found in MCF V2-34. This result could be explained if the non-Akv parents of MCF 247 and MCF V2-34 are different, but could equally well be explained if the 5' end points of recombination differed in these two viruses. [Note that the M-oligonucleotide located at map position 33 in MCF 247 is contained in MCF V2-34 (see Table 2 and Fig. 3A). ] Examination of Table 2 and Fig. 3B gives the impression that the two AKR-derived MCF isolates may be more closely related to each other than to the NIH congenic isolates, which in turn appear to be more closely related to each other. However, it is still possible to envision physical arrangements of the Moligonucleotides in each MCF isolate that would be consistent with identical non-Akv parents for all four isolates. Oligonucleotide maps of MCF 13 and MCF V1-36 may help to resolve
this issue. Correlation of Oligonucleotide Maps with Biological Properties of MCF Viruses and with Tryptic Peptide Anal-
Proc. Natl. Acad. Sci. USA 75 (1978)
499
ysls'of'1Their Virion Properties. Consistent with their dual
(exo/xeno) envelope properties, tryptic peptide analysis of the gp7O proteins of MCF viruses indicates that these proteins contain peptides in common with both ecotropic and xenotropic gp7O proteins (9). The'other virion proteins of MCFs were indistinguishable from those of Akv viruses by tryptic peptide
analysis (9).
Since portions of the Akv genome corresponding to oligonucleotide map positions 21-27 and possibly to the region
between positions 31 and 32 are missing from all four MCFs
(and presumably replaced by non-Akv sequences), RNA sequences in these regions might be involved in specifying host range properties of gp70 and the ability of Akv (and failure of MCFs) to form XC plaques (14). The physical location on the genome of the region between map positions 21 and 27 is estimated to be approximately 1/3 of the genome in from the 3' end. [Oligonucleotides at positions 21-27 are lost or their yield decreased in fingerprints of poly(A)-containing fragmented RNA whose average size is about 106 daltons. ] It is interesting to note a striking similarity between the data in Fig. 3B and oligonucleotide maps of RAV-60 viruses, which are recombinants between exogenous RAV viruses and endogenous information specifying the chf+ phenotype of chicken cells (15). We thank J. W. Hartley and W. P. Rowe for providing the Akv and MCF viruses and for interesting discussions. This work was supported by National Institutes of Health Grant CA-14051 to S. E/ Luria and National Cancer Institute Grant CA-19308 to N.H. J.R. is a Charge de Recherches du Fonds National de la Recherche Scientifique de Belgique and Fellow of the Foundation Rose et Jean Hoguet. 1. Rowe, W. P. (1972) J. Exp. Med. 136, 1272-1285. 2. Rowe, W. P., Hartley, J. W. & Bremner, T. (1972) Science 178, 860-862. 3. Hartley, J. W., Wolford, N. K., Old, 4. J. & Rowe, W. P. (1977) Proc. Natl. Acad. Sci. USA 74, 789-792. 4. Rowe, W. P. & Pincus, T. (1972) J. Exp Med. 135, 429-436. 5. Rommelaere, J., Faller, D. V. & Hopkins, N. (1977) J. Virol. 24, 690-694. 6. Hartley, J. W. & Rowe, W. P. (1975) Virology 65, 128-134. 7. Faller, D. V. & Hopkins, N. (1977) J. Virol. 23, 188-195. 8. Coffin, J. M. & Billeter, M. A. (1976) J. Mol. Biol. 100, 293318. 9. Elder, J., Jensen, F., Lerner, R., Hartley, J. W. & Rowe, W. P. (1977) Proc. Natl. Acad. Sci. USA, 74, 4676-4680. 10. Joho, R. H., Billeter, M. A. & Weissman, C. (1975) Proc. Natl. Acad. Sci. USA 72,4772-4776. 11. Wang, L. H., Duesberg, P. H., Kawai, S. & Hanafusa, H. (1976) Proc. Natl. Acad. Sci. USA 75, 447-451. 12. Wang, L. H., Duesberg, P., Beeman, K. & Vogt, P. K. (1975) J. Virol. 16, 1051-1070. 13. Faller, D. V. & Hopkins, N. (1977) J. Virol. 24, 609-617. 14. Rowe, W. P., Pugh, W. E. & Hartley, J. W. (1970) Virology 42, 1136-1139. 15. Coffin, J. M., Champion, M. A. & Chabot, F. (1977) Proceedings of the ICREW-EMBO Workshop on Avian RNA Tumor Viruses, in press.
Microbiology
Characterization and mapping o RNaseTi-resistant oligonucleotides derived from the genomes of Akv and MCF murine leukemia viruses (recombination)
JEAN ROMMELAERE, DOUGLAS V. FALLER, AND NANCY HOPKINS* Department of Biology and Center for Cancer Research, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139
Communicated by Wallace P. Rowe, October 6, 1977
Ti RNA fingerprints of the genomes of Akv-i ABSTRACT and Akv-2 C-type viruses are indistinguishable and oligonucleotide maps of these viruses are probably the same. Akv-i and -2 share 55-75% of their large Ti-resistant oligonucleotides with four MCF viruses isolated from AKR mice or from NIH Swiss mice that inherit either the Akv-1 or Akv-2 virus-inducing locus of AKR. The majority of Akv oligonucleotides missing from Ti fingerprints of MCFs and the majority of oligonucleotides unique to MCF viruses are clustered and lie at corresponding positions in the 3' half of the oligonucleotide maps of Akv and MCF viruses. The RNA sequences present in different MCF isolates but not present in Akv viruses are related. These results are consistent with a recombinational origin of MCF viruses, as proposed by Hartley and Rowe and their collaborators.
Akv-1 or Akv-2 (1, 2) and MCF (3) viruses can be isolated from mice of the high leukemic AKR strain. Contrary to Akv-1 or -2 viruses, which are present in the mice irrespective of age or tissue and are N-tropic ecotropic viruses (4), MCF viruses are isolated from lymphoid tissue of late preleukemic or leukemic mice, have a dual (exo/xenotropic) host range and other envelope-determined properties, and induce foci in cultured mink cells. To explain the dual host range of MCF viruses and to explain their isolation both from AKR mice and from NIH Swiss mice that inherit either the Akv-1 or Akv-2 virus-inducing locus of AKR, Hartley et al. (3) proposed that MCF viruses may arise in vivo by recombination between an Akv-i or -2 virus and an as yet unidentified xenotropic virus. To investigate this proposal we used Ti RNA fingerprinting and oligonucleotide mapping to analyze the genomes of Akv-i or -2 viruses and of four MCF viruses. Two of the MCF isolates, MCF 13 and MCF 247, were obtained from AKR mice, and two, MCF Vl-36 and MCF V2-34, were obtained from NIH Swiss mice inheriting the Akv-1 or Akv-2 locus of AKR, respectively. Since we have already reported that the large Ti-resistant oligonucleotides of Akv-I and -2 viruses are indistinguishable (5), these viruses will be referred to indiscriminately as Akv viruses where appropriate. MATERIALS AND METHODS Cells and Viruses. The origin of Akv-I and Akv-2 viruses has been described (1, 2, 5). MCF viruses were obtained from old preleukemic AKR mice (MCF 13 and MCF 247) or leukemic. NIH Swiss mice inheriting the Akv-1 (MCF V1-36) or Akv-2 (MCF V2-34) virus-inducing locus of AKR and were purified by successive end-point dilutions. Akv and MCF viruses were kindly provided by J. W. Hartley and W. P. Rowe. SC-i cells (6) chronically infected with each virus were used to prepare 32P-labeled viral RNA. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertsement" in accordance with 18 U. S. C. §1734 solely to indicate
this fact.
Ti RNA Fingerprinting and Oligonucleotide Mapping. Procedures for preparation of 32P-labeled viral RNA, Ti RNA fingerprinting, quantitative and qualitative visual analyses of the products of pancreatic RNase digestion of Ti oligonucleotides (7), and oligonucleotide mapping (refs. 8 and 12; Faller and Hopkins, unpublished data) have been described in detail. The following limitations of the methods of RNA fingerprinting and oligonucleotide mapping should be recalled: (i) Only approximately 5% of the viral genome is contained in the analyzed Ti-resistant oligonucleotides (8); thus critical regions of the genomes may go undetected. (ii) The position of an oligonucleotide on the oligonucleotide maps should in general only be considered accurate to ±1-2 oligonucleotides towards the 3' end of the genome and +2-3 towards the 5' end. (iii) An oligonucleotide map reveals relative, not absolute, positions between Ti oligonucleotides and should only be considered an approximate physical map of the viral genome (see ref. 8).
RESULTS Akv Viruses Share Approximately 55-75% of Their Large RNase Ti-Resistant Oligonucleotides with MCF 13, MCF 247, MCF Vi-36, and MCF V2-34. SC-I cells chronically infected with Akv-i, Akv-2, MCF 13, MCF 247, MCF V1-36, and MCF V2-34 viruses were used to prepare uniformly 32P-labeled 70S viral RNA. The RNA was digested with RNase T1, and the
resulting oligonucleotides were separated by two-dimensional gel electrophoresis. Autoradiograms of the second dimension gels, termed T1 fingerprints, are shown in Fig. 1 A and B for Akv-2 and MCF 247 viruses and diagrams of these fingerprints are shown in Fig. 1 C and D. Diagrams of the T1 fingerprints of MCF 13, MCF VI-36, and MCF V2-34 are shown in Fig. 2.
That the arbitrarily numbered spots in Figs. 1 C and D and 2 represent oligonucleotides derived from the viral genome is inferred from the facts that T1 fingerprints of 70S viral RNA were indistinguishable from those of 35S RNA (data not shown) and that the numbered oligonucleotides are (with two exceptions in MCF VI-36, see legend to Fig. 2) present in molar amount relative to one another in the fingerprints. Molarity of oligonucleotides was determined by quantitation of the radioactivity present in each oligonucleotide in the fingerprints (data not shown) combined with a determination of their length as described below. Since oligonucleotides that differ in sequence or base composition can possess similar electrophoretic mobilities in this system, to ascertain which oligonucleotides of Akv viruses are also present in T1 fingerprints of MCF viruses we further analyzed oligonucleotides of Akv and MCF viruses that appear to have the same electrophoretic mobility. Ti oligonucleotides *
495
To whom reprint requests should be addressed.
496
Proc. Natl. Acad. Sci. USA 75 (1978)
Microbiology: Rommelaere et al.
* * 0
-I'
0 0
000 64?
Ak
A
0
Akv-2
MCF 247
B
0
00~~
2
Akv-2
MCF
247
----------------I
FIG. 1. Two-dimensional gel electrophoretic T1 fingerprints of 70S RNA of Akv-2 and MCF 247 viruses. T1 fingerprints of (A) Akv-2 70S [32P]RNA (2.8 X 106 cpm) and (B) MCF 247 70S [32P]RNA (1.7 X 106 cpm). Autoradiographs were exposed for 26 (Akv-2) and 44 (MCF) hr. (C and D) Diagrams of A and B, respectively. Spot numbers are arbitrary. Open circles in C represent oligonucleotides that are present in molar amount relative to one another. Open circles in D represent single oligonucleotides that are common to Akv and MCF. Black circles (D) represent single oligonucleotides that are found in the fingerprint of MCF but not Akv. Circles that are half white and half black represent spots that consist of two oligonucleotides that comigrate, one of which is probably present in Akv and one of which is MCF-specific. Areas or spots that do not contain a single molar oligonucleotide are cross-hatched. XC and B indicate positions of dye markers xylene cyanol FF and bromphenol blue, respectively. Arrows indicate direction of migration of first and second electrophoresis.
removed from the second dimension gels and digested with pancreatic RNase and the products were separated as described (6). In addition to providing identification of each oligonucleotide, quantitation of these secondary digestion products allowed a determination of their length (h15%) and, in conjunction with quantitation of the radioactivity present in each oligonucleotides in each fingerprint, allowed a determination of their relative molarities. The results of the quantitative analysis of the products of pancreatic RNase digestion of T1 oligonucleotides of Akv and MCF viruses are shown in Table 1 (and ref. 5). These data (or in some cases, qualitative analysis of the secondary digestion products) provide the justification for the diagrams in Figs. 1 C and D and 2. In these diagrams, T1 oligonucleotides that are common to Akv viruses and to a particular MCF virus are represented by open circles. Oligonucleotides that are found in MCF viruses but not in Akv viruses are represented by black circles and are numbered 101, 102, etc. The latter will be referred to as MCF-specific or Moligonucleotides. Of the 41 large Tl-resistant oligonucleotides of Akv viruses that were analyzed, MCF 13 possesses 22/41 or 54%; MCF 247, 30/41 or 74%; MCF V1-36, 29/41 or 70%; and MCF V2-34, 23/41 or 56%. Eight Akv oligonucleotides are not present in any of the four MCF isolates. Of the 25 analyzable M-oligonucleotides in the four MCF viruses (Table 2), one, possibly two, are shared by all four MCF isolates; two, possibly three, are were
shared by three of the four isolates; and six are shared by two of the four viruses. Oligonucleotide Maps of Akv-1, Akv-2, MCF 247, and MCF V2-34. If MCF viruses arise by recombination, then one might expect that M-oligonucleotides and also the Akv oligonucleotides that are missing in T1 fingerprints of each MCF virus would be derived from one or just a few (corresponding) regions of the viral genomes. In particular, since MCF viruses possess altered gp7O proteins relative to Akv viruses (9), and since genetic evidence indicates that gp7O coding sequences of avian (10, 11) and murine (Faller and Hopkins, unpublished data) C-type viruses lie towards the 3' end of the viral RNA, one might expect that M-oligonucleotides would be derived from this region of the MCF genomes. To investigate this possibility we determined the physical order of the T1 oligonucleotides of Akv-i, Akv-2, MCF 247, and MCF V2-34 viruses by determining their relative distances from the 3' ends of the viral RNAs. The methodology used to construct the oligonucleotide maps was similar to that established by Coffin and Billeter (8) and by Wang et al. (12) and identical to that of Faller and Hopkins (unpublished data). The method involves determination of the order in which oligonucleotides are lost from T1 fingerprints of increasingly smaller size classes of poly(A)containing viral RNA. The oligonucleotide maps of Akv-i, MCF 247, and MCF V2-34 are shown in Fig. 3A. [A "crude" map of Akv-2 was obtained from a single experiment (data not
Microbiology: Rommelaere et al.
Proc. Natl. Acad. Sci. USA 75 (1978)
497
-Table 1. Products of pancreatic RNase digestion of large T1 oligonucleotides of Akv and MCF viruses
Oligonucleotide no.* (A)
36 37 38 38a 39 42
(B)
101 102 103 104 105 106 107 110
44
111 112 113 114
(C) (D)
(E)
FIG. 2. Schematic representation of T1 fingerprints of (A) MCF 13 70S [32P]RNA, (B) MCF V1-36, and (C) MCF V2-34. Symbols as in Fig. 1D. [Note that oligonucleotides 1 and 30 of MCF V1-36 are present in less than molar amount, implying that this virus is a mixture of two viruses differing at their 5' end (see Fig. 3A).]
shown) and suggested that corresponding Ti oligonucleotides of Akv-1 and Akv-2 occupy the same relative positions on the genomes of these viruses. ] Not all the Akv or M-oligonucleotides that were analyzed (Table 1 and ref. 5) could be mapped, since, in general, only the order of oligonucleotides that are well separated in a fingerprint can be determined accurately. In some cases, it became possible to map an Akv oligonucleotide that did not possess a unique
115 116 119 120 37 + 37a 38 + 38a 39 + 39a 44+47 3 + 109 9+ 108 12 + 122 35 + 121 107 + 107a 123 + 123a
Composition
5U,1OC,(AU),(A2C),(A2U),(A2G) 2U,7C,3(AC),(AU),G 5U,7C,(AC),(AU),(A2U),G 8U,7C,3(AC),(AG) U,5C,3(AC),(AU),(A3G) 2U,7C,3(AC),(AG) 4U,2C(A2C),(A3C),(A4C) 3U,7C,(AC),(AU),(A2C),(A2U),(A3U),(AG) 4U,1OC,(AC),2(A2C),G 11U,7C,3(AC),(AG) 7U,6C,(AC),(A2U),(A2G) 5U,8C,(AC),G 2U,5C,3(AC),(A4C),G 5U,C,(AU),(A4U),G 2U,10C,(AC),(A2C),G 4U,9C,(AC),(A2C),(AG) 5U,7C,(AC),(AU),(A2U),(A4G) 7U,6C,2(AC),2(AU),(A2C),G 9U,3C,2(AC),2(AU),2(A2U),G
U,3C,(AC),(AU),(A2C),(A3U),(A4C),(A4G) 2U,9C,2(AC),(A2C),G 1OU,5C,2(AC),(AU),(A2U),G 4U,4C,(AC),(AU),(A2C),G 5U,13C,4(AC),(AU),2(A2C),(A2U),G(AG) 11U,13C,3(AC),2(AU),(A2U),G,(AG) 2U,8C,5(AC),(AU),(A2U),(A3G),(A4G) 6U,7C,2(AC),4(AU),(A2C),(A3C),G,(A4G) 6U,16C,2(AC),2(AU),2(A4C),G,(A2G) 13U,11C,3(AC),2(AU),2(A2C),(A2U),(A3C), G,(AG) 6U,23C,(AC),3(AU),(A3C),(AG),G 6U,12C,2(AC),(AU),2(A2C),(AG),G
9U,7C,2(AU),(A4U),2G 5U,7C,2(AC),7(AU),(A3G),G
Average values of at least two determinations, normalized to the yield of the G-containing digestion product and rounded to the nearest integer are given. Approximately 200 cpm per nucleotide were typically found (from 20 X 106 cpm of 70S viral RNA). When the products of pancreatic RNase digestion of homologous oligonucleotides of Akv and/or MCF viruses were indistinguishable (spots that have the same number in the diagrams in Figs. 1 and 2), they are listed only once, or not at all if the products of the Akv oligonucleotide were reported previously (5). (A) Oligonucleotides with unique electrophoretic mobilities in fingerprints of (an) MCF but not an Akv virus (digestion products contained among products of corresponding Akv doublet or complex region); (B) M-oligonucleotides with unique electrophoretic mobilities; (C) oligonucleotides that do not possess unique electrophoretic mobilities in fingerprints of Akv (or MCF); (D) oligonucleotides of MCF that do not possess unique electrophoretic mobilities, one component also present in Akv; and (E) M-oligonucleotides that do not possess unique electrophoretic mobilities. * See diagrams, Figs. 1 and 2.
electrophoretic mobility in fingerprints of Akv viruses because the contaminating Akv oligonucleotide(s) was missing in the fingerprint of one of the two MCF viruses whose T1 oligonucleotides were ordered. The three oligonucleotide maps shown (Fig. 3A) are consistent with the assumption that oligonucleotides that are shared by Akv and MCF viruses occupy the same relative positions on the genomes of these viruses. Thus, to oh. tain a more complete Akv map, we took the libery of pooling the data pertaining to Akv oligonucleotides that were obtained from all the mapping experiments and constructed an oligonucleotide map of Akv referred to as the "pooled data" map
498
Proc. Natl. Acad. Sci. USA 75 (1978)
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Table 2. Distribution of MCF-specific T1 oligonucleotides among four MCF viruses M-Oligonucleotide Cq 0
M
o
+ +
+
+
4
MCF MCF MCF MCF
13 247 V1-36 V2-34
+
l 0
0
+ +
+
+
+
+
LO
?* +
t-
to 0
0
0
+ +
+ +
+
,4 00 C1 0 M M 0 0 vo o '-4 "+ +
u LO
Nil
,
O
,-4
r
4
+
+
+
? ?
+ +
+
0 Cq
H. Cq
Cq
Cq
C Cq
et Cq
Cq
+ +
+
+
+
+
?
e
C1
+
+ +
+
+
+
+
List of MCF-specific oligonucleotides analyzed in all four MCF isolates; their presence in each isolate is indicated by a +. * An oligonucleotide is present at the corresponding electrophoretic position but does not possess a unique electrophoretic mobility and its products of pancreatic RNase digestion are insufficiently distinctive compared to those of the contaminating oligonucleotide(s) to allow unequivocal identification.
(Fig. 3 B and C). Using this map (Fig. 3B) we then identified Akv oligonucleotides that are missing in T1 fingerprints of each MCF virus. These oligonucleotides are indicated by black circles in Fig. 3B. It is apparent that the majority of Akv oligonucleotides that are missing in each MCF virus are clustered on the Akv genome and that the region between map positions 21 and 27 is missing in all four MCF viruses. In Fig. 3C we have indicated the relative positions (on the Akv pooled data map) of those M-oligonucleotides of MCF 247 and MCF V2-34 that could be mapped (black triangles). It is apparent from Fig. 3C (and Fig. 3A) that the majority of these M-oligonucleotides are clustered and lie in the regions of the maps corresponding to those "vacated" by the Akv oligonucleotides.
DISCUSSION We used T1 RNA fingerprinting and oligonucleotide mapping to investigate genetic relationship between Akv and MCF viruses. Our data indicate (i) that the four MCF viruses studied share between 55 and 75% of their T1 oligonucleotides with Akv viruses; (ii) that each MCF isolate is unique; (iii) that the Akv oligonucleotides missing in MCF viruses lie in the 3' half of the Akv oligonucleotide map; and (iv) that the MCF-specific RNA
A
Akv MCF 247 MCF V2-34
sequences present in the four MCF isolates are related and, in the two MCF viruses whose oligonucleotide maps were constructed, these sequences lie at positions on the MCF genomes corresponding to the regions of the missing Akv T1 oligonucleotides. That four MCF isolates differ would seem to support the suggestion that MCFs are not integrated in the mouse genome but arise by recombination involving an Akv virus (3). It is interesting to consider the data further in terms of this proposal. Clusters of M-Oligonucleotides and of Akv Oligonucleotides Missing in MCFs. The majority of M-oligonucleotides present in and the majority of Akv oligonucleotides missing from each MCF isolate are clustered on the oligonucleotide maps of MCF 247, MCF V2-34, and Akv (Fig. 3). (Note, however, that we cannot be certain that these clusters are uninterrupted.) Several explanations can be invoked to explain the presence of black spots and triangles (Fig. 3 B and C) outside these clusters: (i) MCF 13, MCF 247, and MCF V2-34 result from multiple cross-overs between Akv and a virus(es) that shares few or no Ti oligonucleotides with Akv. (ii) The nonAkv, putative xenotropic, parents of MCF viruses share some Ti oligonucleotides with Akv viruses and one or two cross-over events can result in the patterns observed in Fig. 3 B and C. (iii) The inaccuracies inherent in the method of oligonucleotide
5' (1-30) *21- (2-12-15) *27-16-29-5-7-24-4-6-23-28-20-17-25- (11-10) *3- (9-8) -2235- (19-18) *33-26-14 (1-21-30)*2-2- (27-15) -fi1-29 (^65)*7- (438- (37)*6) 4*-1-1 -B08-2- (11-1-19)*33-26-1 (30-1) *1-2- (21-15-V) -39-42-29-4-16-5-1-7-24-4-3 (1- 102-M) *13-15-14-11-26-14
3'
Akv OLIGO. 30( 1-21 21i2)15Q27-39-42(16-4429)(^65) 7-24(37-38- 4- 6)23-282-17-25(u-10) 3- 9- 8-2-35(19 18)33-26-14 1 2 3 4 5 6 7 8 910 2 141516 819 122 231233343537 POSITION
MCF 13
___
B MCF 247
000-000
0
--* ----
MCF V1-36
*--
MCF V2-34
*-..00----..09
MCF 247
v
V
V
0
V
. 0V
V
C MCF V2-34
V
V
V
V
V
V V vV -
FIG. 3. Oligonucleotide maps of Akv-1, MCF 247, and MCF V2-34. (A) Oligonucleotide maps constructed from relative yields of T1 oligonucleotides in fingerprints of fragmented poly(A)-containing RNA of different size classes. Maps of Akv-1, MCF 247, and MCF V2-34 are based on data from, respectively, two, two, and one experiment; eight, seven, and four size classes of RNA were used. Discrepancies between experiments preclude determining the relative order of oligonucleotides within parentheses. MCF-specific oligonucleotides are underlined. Distance between oligonucleotides is arbitrary. (B) "Pooled data" oligonucleotide map (see text) of Akv and diagrams indicating oligonucleotides missing in MCF viruses. The relative positions of 37 Akv T1 oligonucleotides were obtained as described in the text. Absence of an Akv oligonucleotide in T1 fingerprints of MCF viruses is indicated by a black dot; presence of Akv oligonucleotides in MCF viruses is indicated by a line. (C) Relative positions of MCF-specific oligonucleotides (v) on Akv pooled data map. Obtained by comparison of A and B. Spacing between triangles is arbitrary.
Microbiology:
Rommelaere et al.
mapping might explain some of the gaps in Fig. 3 B an uC(rbi example, positions 20, 29, and 36 in MCF 13; see Fig. SB). However, it is unlikely that mapping errors explain the positions outside clusters of the M-oligonucleotides or missing Akv oligonucleotides in MCF 247 or MCF V2-34 (Fig. 3 B and C). (iv) Since single base changes can cause pligonucleotide spots to appear or disappear in a fingerprint (7), it is possible that mutations could explain the location of some of the isolated black spots and triangles in Fig. 3 B and C. However, in those cases where M-oligonucleotides map at positions corresponding to missing isolated Akv oligonucleotides, where the sequences of the M-oligonucleotides are unrelated to those of the "replaced" Akv oligonucleotides (as revealed by Table 1, or ref. 5) and where the M-oligonucleotide appears in more than one MCF virus, we consider this explanation unlikely (for example, positions 32 and 37 in MCF 247). It is interesting to note that the M-oligonucleotides at map positions 32 and 37 of MCF 247 are not alleles of the Akv oligonucleotides missing from the corresponding positions: MCF 13 possesses both the Akv and the M-oligonucleotides that map at position 32 (Table 2); endogenous ecotropic viruses of BALB/c possess both the Akv and the M-oligonucleotides located at position 37 (13). Assuming that the non-Akv parents of MCFs do not possess the Akv oligonucleotide at position 20, the data in Fig. 3B reveal a preferred region, between positions 20 and 21, for one of the proposed recombination events generating MCF viruses. However, because of the limitations of the methods of analysis, we cannot conclude that this point is identical in three MCFs (of four, if one includes MCF 13 by assuming correct placement of the Akv oligonucleotide at position 20; see Fig. 3B). The Putative Xenotropic Parents of Different MCF Isolates Are Related. Our data reveal that the non-Akv RNA sequences present in the genomes of different MCF isolates, whether derived from AKR mice or from NIH congenics, are related. The data do not allow us to conclude whether or not these sequences are identical. Of the four clustered M-oligonucleotides represented by black triangles in the oligonucleotide map of MCF 247 (Fig. 3C), at least two, probably three, are also present in MCF V2-34; the most 5' of the four is not found in MCF V2-34. This result could be explained if the non-Akv parents of MCF 247 and MCF V2-34 are different, but could equally well be explained if the 5' end points of recombination differed in these two viruses. [Note that the M-oligonucleotide located at map position 33 in MCF 247 is contained in MCF V2-34 (see Table 2 and Fig. 3A). ] Examination of Table 2 and Fig. 3B gives the impression that the two AKR-derived MCF isolates may be more closely related to each other than to the NIH congenic isolates, which in turn appear to be more closely related to each other. However, it is still possible to envision physical arrangements of the Moligonucleotides in each MCF isolate that would be consistent with identical non-Akv parents for all four isolates. Oligonucleotide maps of MCF 13 and MCF V1-36 may help to resolve
this issue. Correlation of Oligonucleotide Maps with Biological Properties of MCF Viruses and with Tryptic Peptide Anal-
Proc. Natl. Acad. Sci. USA 75 (1978)
499
ysls'of'1Their Virion Properties. Consistent with their dual
(exo/xeno) envelope properties, tryptic peptide analysis of the gp7O proteins of MCF viruses indicates that these proteins contain peptides in common with both ecotropic and xenotropic gp7O proteins (9). The'other virion proteins of MCFs were indistinguishable from those of Akv viruses by tryptic peptide
analysis (9).
Since portions of the Akv genome corresponding to oligonucleotide map positions 21-27 and possibly to the region
between positions 31 and 32 are missing from all four MCFs
(and presumably replaced by non-Akv sequences), RNA sequences in these regions might be involved in specifying host range properties of gp70 and the ability of Akv (and failure of MCFs) to form XC plaques (14). The physical location on the genome of the region between map positions 21 and 27 is estimated to be approximately 1/3 of the genome in from the 3' end. [Oligonucleotides at positions 21-27 are lost or their yield decreased in fingerprints of poly(A)-containing fragmented RNA whose average size is about 106 daltons. ] It is interesting to note a striking similarity between the data in Fig. 3B and oligonucleotide maps of RAV-60 viruses, which are recombinants between exogenous RAV viruses and endogenous information specifying the chf+ phenotype of chicken cells (15). We thank J. W. Hartley and W. P. Rowe for providing the Akv and MCF viruses and for interesting discussions. This work was supported by National Institutes of Health Grant CA-14051 to S. E/ Luria and National Cancer Institute Grant CA-19308 to N.H. J.R. is a Charge de Recherches du Fonds National de la Recherche Scientifique de Belgique and Fellow of the Foundation Rose et Jean Hoguet. 1. Rowe, W. P. (1972) J. Exp. Med. 136, 1272-1285. 2. Rowe, W. P., Hartley, J. W. & Bremner, T. (1972) Science 178, 860-862. 3. Hartley, J. W., Wolford, N. K., Old, 4. J. & Rowe, W. P. (1977) Proc. Natl. Acad. Sci. USA 74, 789-792. 4. Rowe, W. P. & Pincus, T. (1972) J. Exp Med. 135, 429-436. 5. Rommelaere, J., Faller, D. V. & Hopkins, N. (1977) J. Virol. 24, 690-694. 6. Hartley, J. W. & Rowe, W. P. (1975) Virology 65, 128-134. 7. Faller, D. V. & Hopkins, N. (1977) J. Virol. 23, 188-195. 8. Coffin, J. M. & Billeter, M. A. (1976) J. Mol. Biol. 100, 293318. 9. Elder, J., Jensen, F., Lerner, R., Hartley, J. W. & Rowe, W. P. (1977) Proc. Natl. Acad. Sci. USA, 74, 4676-4680. 10. Joho, R. H., Billeter, M. A. & Weissman, C. (1975) Proc. Natl. Acad. Sci. USA 72,4772-4776. 11. Wang, L. H., Duesberg, P. H., Kawai, S. & Hanafusa, H. (1976) Proc. Natl. Acad. Sci. USA 75, 447-451. 12. Wang, L. H., Duesberg, P., Beeman, K. & Vogt, P. K. (1975) J. Virol. 16, 1051-1070. 13. Faller, D. V. & Hopkins, N. (1977) J. Virol. 24, 609-617. 14. Rowe, W. P., Pugh, W. E. & Hartley, J. W. (1970) Virology 42, 1136-1139. 15. Coffin, J. M., Champion, M. A. & Chabot, F. (1977) Proceedings of the ICREW-EMBO Workshop on Avian RNA Tumor Viruses, in press.
