Cell, Vol. IO. 649-657
April
1977, Copyright
0 1977 by MIT
Chicken Macrochromosomes Contain an Endogenous Provirus and Microchromosomes Contain Sequences Related to the Transforming Gene of ASV Thomas G. Padgett, Elton Stubblefield* Harold E. Varmus Department of Microbiology University of California San Francisco, California 94143
and
* Department of Cell Biology University of Texas Cancer Center Houston, Texas 77025 Summary Chicken chromosomes from a euploid Marek’s lymphoma cell line have been partially fractionated according to size by rate zonal centrifugation in a zonal rotor. DNA-DNA hybridization tests, using unlabeled DNA extracted from gradient fractions and labeled single-stranded, virus-specific DNAs prepared in vitro, indicate that large macrochromosomes harbor the provirus for the endogenous RNA tumor virus of chickens (RAV0), whereas a cellular sequence related to the transforming gene of avian sarcoma virus (ASV) is located in microchromosomes. In support of the method, we have also shown that the single gene for ovalbumin can be assigned to macrochromosomes. Introduction Physical mapping of genes present in low numbers of copies in eucaryotic genomes has lagged behind genetic analysis using somatic cell hybrids (Davidson and de la Cruz, 1974). However, the combination of large-scale chromosomal fractionation procedures and molecular hybridization techniques capable of measuring single-copy genes now permit assignment of such genes to a portion of a eucaryotic genome, if not to a specific chromosome. We have used this combination of technical advances to ask whether nucleotide sequences related to genetically defined components of avian sarcoma viruses (ASV) are physically linked in normal chicken DNA. The RNA genome of ASV is currently believed to contain four genes (Baltimore, 1975; Figure 1) which have been provisionally mapped by chemical and genetic studies (Duesberg et al., 1976; Joho et al., 1976). One gene, designated src, is not essential for viral replication, but is required for sarcomagenesis in vivo and for transformation of fibroblasts in vitro. The other three genes, designated gag, env and pal, are necessary for viral replication and code for viral core proteins (group-specific antigens), envelope glycoproteins and the viral RNA-directed DNA polymerase, respectively. Certain normal chicken cells can spontaneously pro-
duce RAV-0, an apparently nonpathogenic virus closely related to ASV by nucleic acid hybridization tests (Vogt and Friis, 1971; Neiman et al., 1974; Shoyab and Baluda, 1975). Although RAV-0 RNA contains sequences necessary for viral replication similar to those found in ASV RNA, it appears to lack sequences present in the src gene, since a hybridization reagent (cDNA~,,) specific for most or all of the src gene does not hybridize to RAV-0 RNA (Stehelin et al., 1976a). RAV-O-related sequences are found endogenously in the chromosomal DNA of all chickens tested, but little if any of the RAV-0 genome is present in the DNA of other avian species (Neiman, 1973; Kang and Temin, 1974; Varmus, Heasley and Bishop, 1974; Tereba, Skoog and Vogt, 1975). Normal cell DNA from all avian species, however, contains one or two copies per haploid genome of sequences which anneal to cDNA,,,, (“sarc” sequences) (Stehelin et al., 1976b). The “sarc” sequences are diverged among avian species in accord with described evolutionary relationships, with the chicken “sarc” sequences being the most closely related to the src gene present in ASV RNA. The presence of DNA related to both the replicative and the transforming genes of ASV in normal chicken cells supports the possibility that ASV may have arisen from those components by a mechanism as yet unknown (Temin, 1974; Martin and Weiss, 1974; Varmus et al., 1976). TO examine the physical relationship between the two ASV-related components of chicken DNA, we have used molecular hybridization to locate the ASV-related sequences in chicken chromosomes fractionated by rate zonal sedimentation in neutral sucrose gradients. In such gradients, the 50-60 indistinguishable avian microchromosomes were effectively separated from the 14 pairs of macrochromosomes, and partial fractionation of the macrochromosomes was also achieved, as determined by analysis with flow microfluorimetry. Analysis of fractionated chromosomal DNA with hybridization reagents specific for RAV-O-related (replicative) sequences indicates that these sequences are in the largest class of macrochromosomes. In contrast, cDN$,,, annealed principally with the DNA extracted from the microchromosomes, indicating that “sarc” sequences are physically separated from RAV-0 proviral sequences in the chicken genome. In addition, the method has been validated by locating the ovalbumin gene in macrochromosomes. Results Characterization of MSB-1 Cells In the studies reported here, chromosomes were prepared from a lymphoblastoid cell line (MSB-1)
Cell 650
gag
5’
,
PO1
I
+ ’
CORE PROTEINS
1
I
en”
Figure
1. Provisional of the genes et al. (1976).
Genetic
3’
’ + ’
ENVELOPE GLY COPROTEINS
REVERSE TRANSCRIPTASE
The order and Joho
src I
1 TRANSFORMING PROTEIN (?)
Map of ASV
is as suggested
by Duesberg
et al. (1976)
established from a chicken infected with Marek’s disease virus (Akiyama and Kato, 1974; Nazerian and Lee, 1974). MSB-1 cells were used for these analyses for several reasons: first, their short doubling time (8-12 hr) allows up to 50% of the cells to accumulate in metaphase during a 6 hr exposure to colcemid; second, the karyotype of MSB-1 cells is virtually identical to the normal chicken karyotype, differing only by two small translocations; third, the cells grow in suspension, facilitating the preparation of the large number of chromosomes required for molecular hybridization; and fourth, these cells have not been infected by and are not producing avian RNA tumor viruses. The karyotype of MSB-1 lymphoblasts (Figure 2) is typical of normal avian cells (Tagaki and Sasaki, 1974), containing 28 macrochromosomes (13 pairs of large autosomal chromosomes and the sex chromosomes Z and W) and approximately 60 indistinguishable microchromosomes. The only detectable abnormalities are small translocations on the largest macrochromosome (Iq+) and on a sex chromosome (Wp+); the source of the translocated DNA is not known. The karyotype of the MSB-1 line has remained stable since isolation of the cell line in 1973. Several tests were performed to substantiate our presumption that the MSB-1 cells had not been inadvertantly infected by avian leukosis or sarcoma viruses, since we wished to measure only endogenous virus-specific DNA in the fractionated chromosomes. -Undiluted culture medium from MSB-1 cells showed no focus-forming activity on C/E chicken cells. Infection of MSB-1 cells with ASV led to the production of high titers of focus-forming virus (up to 3 x lo6 focus-forming units per ml), indicating that the cells are permissive for replication of chicken viruses. -To test for production of nontransforming as well as transforming viruses, medium from MSB-1 cultures was examined for virus-specific RNA in sedimentable particles by molecular hybridization with according to the method described by =P-cDNA,,,, Ringold et al. (1975). No hybridization was observed, indicating that
April
1977, Copyright
0 1977 by MIT
Chicken Macrochromosomes Contain an Endogenous Provirus and Microchromosomes Contain Sequences Related to the Transforming Gene of ASV Thomas G. Padgett, Elton Stubblefield* Harold E. Varmus Department of Microbiology University of California San Francisco, California 94143
and
* Department of Cell Biology University of Texas Cancer Center Houston, Texas 77025 Summary Chicken chromosomes from a euploid Marek’s lymphoma cell line have been partially fractionated according to size by rate zonal centrifugation in a zonal rotor. DNA-DNA hybridization tests, using unlabeled DNA extracted from gradient fractions and labeled single-stranded, virus-specific DNAs prepared in vitro, indicate that large macrochromosomes harbor the provirus for the endogenous RNA tumor virus of chickens (RAV0), whereas a cellular sequence related to the transforming gene of avian sarcoma virus (ASV) is located in microchromosomes. In support of the method, we have also shown that the single gene for ovalbumin can be assigned to macrochromosomes. Introduction Physical mapping of genes present in low numbers of copies in eucaryotic genomes has lagged behind genetic analysis using somatic cell hybrids (Davidson and de la Cruz, 1974). However, the combination of large-scale chromosomal fractionation procedures and molecular hybridization techniques capable of measuring single-copy genes now permit assignment of such genes to a portion of a eucaryotic genome, if not to a specific chromosome. We have used this combination of technical advances to ask whether nucleotide sequences related to genetically defined components of avian sarcoma viruses (ASV) are physically linked in normal chicken DNA. The RNA genome of ASV is currently believed to contain four genes (Baltimore, 1975; Figure 1) which have been provisionally mapped by chemical and genetic studies (Duesberg et al., 1976; Joho et al., 1976). One gene, designated src, is not essential for viral replication, but is required for sarcomagenesis in vivo and for transformation of fibroblasts in vitro. The other three genes, designated gag, env and pal, are necessary for viral replication and code for viral core proteins (group-specific antigens), envelope glycoproteins and the viral RNA-directed DNA polymerase, respectively. Certain normal chicken cells can spontaneously pro-
duce RAV-0, an apparently nonpathogenic virus closely related to ASV by nucleic acid hybridization tests (Vogt and Friis, 1971; Neiman et al., 1974; Shoyab and Baluda, 1975). Although RAV-0 RNA contains sequences necessary for viral replication similar to those found in ASV RNA, it appears to lack sequences present in the src gene, since a hybridization reagent (cDNA~,,) specific for most or all of the src gene does not hybridize to RAV-0 RNA (Stehelin et al., 1976a). RAV-O-related sequences are found endogenously in the chromosomal DNA of all chickens tested, but little if any of the RAV-0 genome is present in the DNA of other avian species (Neiman, 1973; Kang and Temin, 1974; Varmus, Heasley and Bishop, 1974; Tereba, Skoog and Vogt, 1975). Normal cell DNA from all avian species, however, contains one or two copies per haploid genome of sequences which anneal to cDNA,,,, (“sarc” sequences) (Stehelin et al., 1976b). The “sarc” sequences are diverged among avian species in accord with described evolutionary relationships, with the chicken “sarc” sequences being the most closely related to the src gene present in ASV RNA. The presence of DNA related to both the replicative and the transforming genes of ASV in normal chicken cells supports the possibility that ASV may have arisen from those components by a mechanism as yet unknown (Temin, 1974; Martin and Weiss, 1974; Varmus et al., 1976). TO examine the physical relationship between the two ASV-related components of chicken DNA, we have used molecular hybridization to locate the ASV-related sequences in chicken chromosomes fractionated by rate zonal sedimentation in neutral sucrose gradients. In such gradients, the 50-60 indistinguishable avian microchromosomes were effectively separated from the 14 pairs of macrochromosomes, and partial fractionation of the macrochromosomes was also achieved, as determined by analysis with flow microfluorimetry. Analysis of fractionated chromosomal DNA with hybridization reagents specific for RAV-O-related (replicative) sequences indicates that these sequences are in the largest class of macrochromosomes. In contrast, cDN$,,, annealed principally with the DNA extracted from the microchromosomes, indicating that “sarc” sequences are physically separated from RAV-0 proviral sequences in the chicken genome. In addition, the method has been validated by locating the ovalbumin gene in macrochromosomes. Results Characterization of MSB-1 Cells In the studies reported here, chromosomes were prepared from a lymphoblastoid cell line (MSB-1)
Cell 650
gag
5’
,
PO1
I
+ ’
CORE PROTEINS
1
I
en”
Figure
1. Provisional of the genes et al. (1976).
Genetic
3’
’ + ’
ENVELOPE GLY COPROTEINS
REVERSE TRANSCRIPTASE
The order and Joho
src I
1 TRANSFORMING PROTEIN (?)
Map of ASV
is as suggested
by Duesberg
et al. (1976)
established from a chicken infected with Marek’s disease virus (Akiyama and Kato, 1974; Nazerian and Lee, 1974). MSB-1 cells were used for these analyses for several reasons: first, their short doubling time (8-12 hr) allows up to 50% of the cells to accumulate in metaphase during a 6 hr exposure to colcemid; second, the karyotype of MSB-1 cells is virtually identical to the normal chicken karyotype, differing only by two small translocations; third, the cells grow in suspension, facilitating the preparation of the large number of chromosomes required for molecular hybridization; and fourth, these cells have not been infected by and are not producing avian RNA tumor viruses. The karyotype of MSB-1 lymphoblasts (Figure 2) is typical of normal avian cells (Tagaki and Sasaki, 1974), containing 28 macrochromosomes (13 pairs of large autosomal chromosomes and the sex chromosomes Z and W) and approximately 60 indistinguishable microchromosomes. The only detectable abnormalities are small translocations on the largest macrochromosome (Iq+) and on a sex chromosome (Wp+); the source of the translocated DNA is not known. The karyotype of the MSB-1 line has remained stable since isolation of the cell line in 1973. Several tests were performed to substantiate our presumption that the MSB-1 cells had not been inadvertantly infected by avian leukosis or sarcoma viruses, since we wished to measure only endogenous virus-specific DNA in the fractionated chromosomes. -Undiluted culture medium from MSB-1 cells showed no focus-forming activity on C/E chicken cells. Infection of MSB-1 cells with ASV led to the production of high titers of focus-forming virus (up to 3 x lo6 focus-forming units per ml), indicating that the cells are permissive for replication of chicken viruses. -To test for production of nontransforming as well as transforming viruses, medium from MSB-1 cultures was examined for virus-specific RNA in sedimentable particles by molecular hybridization with according to the method described by =P-cDNA,,,, Ringold et al. (1975). No hybridization was observed, indicating that
