Four male domestic cats were selected for FISH mapping based on the geographic diversity of their origins or on the presence in their genomes of two previously characterized enFeLV proviruses (50
) (Table ). The four cats selected included a Burmese cat (Fca-9), representing a breed of Southeast Asian origin (32
); an Egyptian Mau cat (Fca-146), part of a breed likely derived from Egyptian cats (32
); a Persian cat (Fca-215), of a breed derived from cats in Iran (32
); and a nonbreed cat from the United States (Fca-127) (Table ).
Domestic cats selected for enFeLV FISH
A 7-kb DNA template of endogenous FeLV, designated 80(enFeLV-GGAG)-gpe, was constructed and used as a probe for FISH. The DNA fragment included the gag
, and env
regions of a full-length endogenous FeLV (50
). The probe template excluded the proviral LTRs in order to preclude detection of ca. 150 copies of enFeLV LTRs without associated coding regions that are present in the genome (6
). The presence of exogenous FeLV proviruses in the genomes of the four cats was ruled out by PCR screening (see Materials and Methods).
FISH images of metaphase chromosomes and proviral signals are shown in Fig. . Twenty-nine distinct enFeLV autosomal loci were identified across the four cats. The number of autosomal enFeLV loci detected in each cat ranged between 9 and 16 (Table and Fig. and ). The Persian cat, Fca-215, had 13 autosomal enFeLV loci: 2 distinct integration sites in chromosome A2, 2 in chromosome B1, 1 in B2, 1 in B3, 3 in B4, 1 in C1, 1 in D4, 1 in E1, and 1 in F1 (Fig. and ). The Egyptian Mau cat, Fca-146, had 16 distinct autosomal enFeLV loci: 3 in chromosome A1, 1 in B1, 1 in B2, 2 in B3, 3 in B4, 2 in D2, 1 in D4, 2 in E3, and 1 in F1 (Fig. and ). The nonbreed cat, Fca-127, had nine distinct autosomal enFeLV loci: two in chromosome A2, one in B1, one in B2, two in B3, two in B4, and one in D4 (Fig. and ). The Burmese cat, Fca-9, had 13 distinct autosomal enFeLV loci: 2 in chromosome A2, 2 in B1, 1 in B2, 2 in B3, 4 in B4, and 2 in C1 (Fig. and ).
FIG. 1. Chromosomal localization of enFeLVs in metaphase spreads from four domestic cats visualized by FISH. The probe consisted of a segment of enFeLV including gag, pol, and env without LTRs. In panels a to d, hybridization signals (red) are superimposed on (more ...)
FIG. 2. Ideogram (10) summarizing chromosomal locations of enFeLV copies as determined by FISH in four domestic cats; a distinct icon shape represents each cat. The icons represent loci at which enFeLV was detected in at least 10% of the metaphase chromosome (more ...)
The number and location of enFeLVs detected by FISH in the four cats are summarized to the left of each cat chromosome in the ideogram (10
) in Fig. , which positions the 29 distinct autosomal enFeLV integration loci observed across the four cats. Five of these sites (B1q36, B2p15, B3q25, B4q11, and B4q15) were common to all four cats. At 24 other autosomal loci, signal was present only for three (2 sites), two (3 sites), or one (19 sites) of the four cats (Fig. ); the unique sites limited to one of the four cats are also summarized in Table . No enFeLV loci were detected for any of the four cats in chromosome A3, C2, D1, D3, E2, or F2. An X chromosome enFeLV locus was found only in the Burmese cat, Fca-9 (Fig. , ). Of the 30 enFeLV loci in the X chromosome and autosomes, 10 were telomeric, 1 each in A2p, B1q, B2p, B3q, B4p, B4q, C1p, E1q, E3p, and Xq (Fig. and ). Only two were centromeric, in chromosomes A2 and B4 (Fig. and ). The Y chromosomes in all four cats displayed multiple enFeLV loci (Fig. ). For each cat, enFeLV was detected at one position in the Yp arm, with signal on two to four positions in the Yq arm. An elongated Yq chromosome arm was present in the Persian cat, Fca-215; additional sites positive for enFeLV were detected in this elongated region of the Y chromosome, possibly suggesting duplication of a Yq fragment (Fig. ).
For each cat in Fig. , loci at which enFeLV signal was heterozygous, i.e., present in a single chromosome homologue, and homozygous loci at which signal was present in both chromosome homologues in spreads are indicated. The proportion of homozygous sites (Table and Fig. ) was higher for the Burmese cat, Fca-9 (77% of sites were double), than for the other cats (44% for Fca-127, 37% for Fca-146, and 46% for Fca-215). There were a total of 77 enFeLV copies present at the 29 loci across 144 autosomes (2 homologues for each autosome × 18 autosomes per cat × 4 cats). The only autosomal enFeLV locus that was homozygous in all four cats was the site at B4q15 (Fig. ). The total number of copies of enFeLV found across the eight copies of each autosome (4 cats × 2 homologous chromosomes per cat) was greater for the longer chromosomes (R2
= 0.119) (Fig. ) (30
). The eight B4 chromosomes (4 cats × 2 B4 chromosomes per cat) contained a total of 21 copies of enFeLV (Fig. ), which was higher than expected based on chromosomal length (Fig. ) (P
= 0.0063). Since three of the four cats had been selected for FISH based on known enFeLV proviruses that mapped to chromosome A1 or A2, to exclude ascertainment bias, a separate analysis was run excluding chromosomes A1 and A2 (R2
= 0.159; y
− 0.3812; P
= 0.0089 [t
FIG. 3. Copies of endogenous feline leukemia proviruses across four cats versus lengths of autosomes. The x axis indicates the relative physical length of each autosome (30). There were a total of 77 copies of enFeLV across the 144 autosomes examined (4 cats (more ...)
In cat Fca-127, two enFeLV sites in the A2 chromosome homologues were in trans configuration: one of the A2 homologues had a single enFeLV copy at A2p24; the other homologue had a single enFeLV copy at A2q11 (Fig. ). However, ~40% of the metaphase spreads of Fca-127 were trisomic for chromosome A2. In trisomic cells, only the A2q11 signal appeared twice in the same metaphase spread. Thus, A2 trisomy resulted from duplication of the homologue carrying enFeLV at A2q11. Only data from nontrisomic metaphase spreads are included in Fig. and other summaries of the enFeLV FISH results.
An RH panel developed for the domestic cat (35
) was used to map the chromosomal location of the unique cat genomic sequence flanking the integration sites of enFeLV-AGTT (50
), enFeLV-GGAG (50
), and three novel enFeLV proviruses, enFeLV-CTCT, -AGAG, and -ATGC. The genomic DNA flanking the integration site of enFeLV-AGTT mapped to A1q at 1,250.38 centirays (cR5000
) between the loci SEPP1
. The DNA flanking enFeLV-GGAG mapped to A2q at 787.91 cR5000
between loci DDC
. The DNA flanking enFeLV-CTCT mapped to C1p in the interval between loci GJA4
based on two-point logarithm of the odds scores. The flanks of two endogenous FeLVs, enFeLV-AGAG and -ATGC, had high retention frequencies (≥80%) in the RH panel and could not be mapped. High retention frequency is indicative either of a centromeric location or of primers that are amplifying multiple loci (e.g., enFeLV integration within repeat sequences of the feline genome common to multiple chromosomal locations).
Three of the four cats used for FISH mapping were selected in part based upon their carrying previously characterized enFeLV proviruses, enFeLV-AGTT and enFeLV-GGAG (Table ) (50
). PCR screens for specific enFeLVs (with one of the primers based on flanking DNA) had demonstrated the presence of enFeLV-AGTT in Fca-146 (in one of the two chromosome homologues) but not in the other three cats (50
). Consistent with the PCR results, Fca-146 was the only cat for which FISH detected enFeLV signal in chromosome arm A1q (Fig. and ), and RH mapping assigned the enFeLV-AGTT flanking region to A1q. Likewise, enFeLV-GGAG had been detected by PCR (with one of the primers based on flanking DNA) in Fca-127 (one homologue) and Fca-215 (both homologues), but not in the other cats (50
). Consistent with those results, radiation hybrid mapping assigned the enFeLV-GGAG flank to chromosome A2q, and FISH demonstrated the presence at A2q11 of an enFeLV site found in one homologue of Fca-127 and both homologues of Fca-215 (Fig. and ). For both enFeLV proviruses, the results of FISH mapping, RH mapping, and previous PCR amplification (with one of the primers based on flanking DNA) were consistent for the four domestic cats. The enFeLV integrations detected by FISH that correspond to the physically mapped enFeLVs are indicated in Fig. .