key: cord-0004811-8en3vrs9
authors: BENVENISTE, RAOUL E.; TODARO, GEORGE J.
title: Segregation of RD-114 and FeLV-related sequences in crosses between domestic cat and leopard cat
date: 1975
journal: Nature
DOI: 10.1038/257506a0
sha: 23d9759a661ac8679e869223b1c6539c51f7161c
doc_id: 4811
cord_uid: 8en3vrs9

TYPE C viruses of the RD-114 (ref. 1) group have been isolated, either spontaneously or after chemical induction, from cell cultures of the domestic cat (Felis catus)(2–4). Nucleic acid sequences related to the RD-114 genome are in the DNA of all domestic cats(5–8). Thus these viral genomes are transmitted vertically from parent to offspring as integral components of cat cellular DNA. Although the family Felidae consists of closely related animals, only four Felis species have been found to contain RD-114-related sequences. These include the domestic cat, the European wildcat (F. sylvestris), the sand cat (F. margarita), and the jungle cat (F. chaus); other members of the Felidae lack nucleic acid sequences related to RD-114 (ref. 9). The observation that RD-114 is partially related to the endogenous baboon type C viruses(10–12) and that sequences related to RD-114 are found in the cellular DNA of all Old World monkeys led to the postulate that this group of viruses originated from an endogenous primate type C virus(13) transmitted horizontally to the germ line of ancestors of certain Felis species during the Pliocene or early Pleistocene somewhere in the region of the Mediterranean basin(9).

Segregation of RD-114 and FeL V -related sequences in crosses between domestic cat and leopard cat TYPE C viruses of the RD-114 (ref. I) group have been isolated, either spontaneously or after chemical induction, from cell cultures of the domestic cat (Felis catus)2-4. Nucleic acid sequences related to the RD-114 genome are in the DNA of all domestic cats 5 -8. Thus these viral genomes are transmitted vertically from parent to offspring as integral components of cat cellular DNA. Although the family Felidae consists of closely related animals, only four Felis species have been found to contain RD-114-related sequences. These include the domestic cat, the European wildcat (F. sylvestris), the sand cat (F. margarita), and the jungle cat (F. chaus); other members of the Felidae lack nucleic acid sequences related to . The observation that RD-114 is partially related to the endogenous baboon type C viruses 10 -12 and that sequences related to RD-114 are found in the cellular DNA of all Old World monkeys led to the postulate that this group of viruses originated from an endogenous primate type C virus 13 transmitted horizontally to the germ line of ancestors of certain Felis species during the Pliocene or early Pleistocene somewhere in the region of the Mediterranean basin 9 • A second distinct group of type C viruses, the feline leukaemia viruses (FeL V), also has been isolated from domestic cats14. Although FeLVs are horizontally transmitted among domestic cats, genes partially related to the RNA genome of FeL V are found in F. catus DNN5 and in the DNA of the other species of Felidae which contain RD-114 related nucleic acid sequences 16 . Viruses of the FeL V group are also postulated to have been transmitted to an ancestor of these Felis species, but to have originated from a rodent rather than a primate source 16 .

The leopard cat (F. bengalensis) is a spotted wildcat found throughout South-east Asia which lacks RD-114 and FeLVrelated DNA sequences (RD-, FL -)9. Since leopard cats produce viable offspring when bred with domestic cats (RD+, FL +), we studied the segregation of both sets of virogenes in Fl hybrids and in the progeny of a backcross to the RD-, FL -parent. The cellular DNA of the Fl hybrids contains half the number of copies of each set of sequences. The RD and FL virogenes segregate together in the backcrossed animals in a manner consistent with their localisation at a single chromosomal site.

The reassociation kinetics obtained by hybridising 3H-DNA transcripts of viral RNA to cellular DNA can be used to estimate relative gene frequencies by determination of half Cot values (the midpoint of the renaturation curve)!'. The number of gene copies can also be estimated by plotting reassociation kinetics as the reciprocal of the fraction of unhybridised 3H-DNA against Cot (Wetmur-Davidson plot)l8. In such plots, the slope is proportional to the number of copies of those sequences measured. Using RD-114 3H-DNA probes, Nature Vol. 257 October 9 1975 multiple copies of virus-related sequences can be detected in the cellular DNA of stray domestic cats, domestic cats reared in a germ-free environment (Merck, Sharp and Dohme, West Point, Pennsylvania) and in European wildcats (F. sylvestris) ( Fig. la and Table I ). The Cot t values ranged from 120 to 170. In contrast, the cellular DNA of the leopard cat completely lacks RD-114 related sequences. The Cot t values for the selfannealing of non-repetitive domestic cat cellular DNA, and for the hybridisation of the 3H-DNA RD-114 probe to the DNA of a canine thymus cell line infected with RD-114, ranged from 1,800 to 2,000 ( Fig. 1 , Table I ). Given that Cot 1 values of 1,800--2,000 are obtained with genes present in a si~gle copy per haploid genome, domestic cat and European wildcat cellular DNAs contain 10-13 copies of RD-114-related sequences per haploid genome, Since these copies represent a family of diverging gene sequences only partially related to one another 19 , the calculated number of copies may be an underestimate 20 ,21.

Leopard cat males were mated to domestic cat females and the F 1 hybrids studied. These DNAs contain a complete complement of sequences related to the RD-114 probe, but only half the number of copies present in the domestic cat parent (Fig. la) , The C of t values (275-350) represent, as a minimum estimate, five to seven virogene copies per haploid genome. Two kittens obtained from an F 1 hybrid female backcrossed to the leopard cat ( Fig. 2) were also studied. Figure la shows that kitten No.1 contains all the RD-l14-related information, but only half the number of copies (Cot t 280), like the F 1 parent, whereas kitten No.2 (from the same litter) lacks RD-114-related DNA sequences like its leopard cat parent. These results suggest that the multiple copies of RD-114 re- kidney, lung) and hybridised to RD-114 and FeLV 3H-DNA as described in Fig. I . Domestic cats Nos 2 and 3 were from the germfree colony of cats at Merck, Sharp and Dohme; animals from this colony have never been found to be positive for infectious feline leukaemia virus. FI hybrid cats Nos I, 2 and 3 and 4 belong to three separate litters. t C ott values represent the midpoint of the reannealing curves".

:j: The approximate number of copies per haploid genome of sequences related to either RD-114 or FeL V were estimated from reciprocal plots (Fig. I) . The number of copies is determined by the ratio of the slope of each line to the slope of the line described by the reassociation of non-repetitive domestic cat cellular DNA (Cot, = 1,800-2,000; see also ref. 19 ). In the case of RD-114, where two sets of viral sequences can be detected in cellular DNA, the number of copies listed is the average of the two populations. § CCC clone 6 is from a continuous line of domestic cat kidney fibroblasts and is not releasing type C virus"', and RD-114/FCf2Th and FeL V /FCf2Th are a dog thymus cell line infected, respectively, with RD-1I4 (ref. 1) or with the helper virus from the Gardner-Arnstein" strain of feline sarcoma virus. lated sequences are located together at one (or relatively few) chromosomal sites. The same cats were examined for FeLV-related genes using 3H-DNA probes prepared from various strains of FeLV22,23. No cross-hybridisation between these probes and RD-114 is detectable'5.24. The results parallel exactly the data obtained with transcripts of RD-114 RNA. The domestic cat parent contains multiple copies of FeLV-related virogenes whereas the leopard cat parent lacks these sequences. The F I hybrids contain half the number of copies. Backcrossed kitten No.1 has the same number of virogene copies as its parent (the F, hybrid) although its littermate has no detectable sequences related to FeL V. Table 1 summarises the hybridisation data; four clear classes of DNAs are evident. The first consists of animals that contain full complements of RD-114 and Fe LV-related gene sequences. These cats contain both sets of virogene sequences reiterated a comparable number of times, although there may be fewer FeLV-related copies. The animals in the second class, including all the F, hybrids and one of the backcrossed kittens, contain half the virogene complement of the RD+, FL + parents. A third class contains sequences that anneal to the viral probe with reassociation kinetics comparable with that seen for the association of the most slowly reannealing cellular DNA sequences and therefore probably contains one viral copy. This includes cloned heterologous cell lines producing high titres of either RD-114 or FeL V viruses. The fourth class completely lacks sequences related to either RD-114 or FeLV and includes the leopard cats and one of the backcrossed kittens. that each of the multiple copies of RD-1l4 or FeLV-related virogenes occurs on a different linkage group, as well as models of non-chromosomal inheritance of the mUltiple viral copies. Since only certain Felis species contain RD-1l4 and FeLVrelated sequences in their DNA, we propose that both classes of viruses were acquired by cats subsequent to their major radiation, most likely in the Pliocene, and that both sets of sequences have been perpetuated in the germ line 9 ,16. The mUltiple virogene copies presumably arose as a result of gene duplication and/or unequal crossing-over after infection. The presence of multiple copies of both sets of virogenes in cat cellular DNA seems to be a general property of endogenous mammalian type C viruses; mouse, rat, hamster, pig and baboon DNAs also contain similar numbers of copies of their respective endogenous viruses 19 • The genetic crosses described here provide a new approach to the study of the evolution of multiple gene systems. The virogene sequences can be considered as one of the group of moderately repetitive sequences such as the genes for 5S RNA26, histones 26 and feather keratin 20 • The existence of natural populations of animals that either lack or contain DNA sequences related to both RD-1l4 and FeLV, and the ability of these cats to interbreed permits the study of the physiological and potentially pathological role of each of these genetically transmitted gene sequences. Hybrid animals containing half the number of virogene copies and virogenenegative cats should allow the study of the effects of gene dose on susceptibility and resistance to diseases mediated by both groups of type C viruses.

Identification of heat-dissociable RNA complexes in two porcine coronaviruses THE corona virus genome has been shown to comprise singlestranded RNAI". Examination of the viral nucleic acid synthesised by pig kidney cells infected with transmissible gastroenteritis virus (TGEV) suggested that several molecular species, ranging in size between 18 and 28S, were involved in the viral replicative cycle 3 ; similarly Tannock found a wide variation in the size of RNA molecules extracted from avian infectious bronchitis virus (IBV) by a phenol-sodium dodecyl sulphate (SDS) method'. Extraction of IBV RNA by 1 % SDS at 60°C has however revealed a single component of molecular wei~ht 9 X 10: corresponding to 60S by electrophoresis through 2.2 % polyacrylamide gels'.

We have examined the RNA extracted from purified preparations of TGEV and a second porcine coronavirus -haemagglutinating encephalomyelitis virus (HEV)-and have found a 60--70S RNA component which dissociates into 35S and 4S material on heating above 60 °C in a way that closely resembles the genome of the oncogenic RNA viruses.

We had observed that treatment of purified TGEV with 1 % SDS at 20 °C disrupted the virions and liberated a high molecular weight complex containing the RNA. On the assumption that this complex might comprise the hitherto undetected ribonucleoprotein, we extracted the material from TGEV preparations radioactively labelled with 3H_ uridine or with 3H-leucine to determine which structural polypeptide was associated with the complex, As is shown in Fig. I , however, the fast moving RNA complex has no detectable protein associated with it, while polyacrylamide gel analysis of the radioactivity remaining near the top of 

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