Molecular cloning of the feline c-fes proto-oncogene and construction of a chimeric transforming gene

The feline c-fes proto-oncogene, different parts of which were captured in feline leukemia virus (FeLV) to generate the transforming genes (v-fes) of the Gardner-Arnstein (GA) strain of feline sarcoma virus (FeSV) and the Snyder-Theilen strain (ST) of FeSV, was cloned and its genetic organization determined. Southern blot analysis revealed that the c-fes genetic sequences were distributed discontinuously and colinearly with the v-fes transforming gene over a DNA region of around 12.0 kb. Using cloned c-fes sequences, complementation of GA-FeSV transforming activity was studied. Upon replacement of the 3' half of v-fesGA with homologous feline c-fes sequences and transfection of the chimeric gene, morphological transformation was observed. Immunoprecipitation analysis of these transformed cells revealed expression of high Mr fusion proteins. Phosphorylation of these proteins was observed in an in vitro protein kinase assay, and tyrosine residues appeared to be involved as acceptor amino acid.     

Molecular cloning of Snyder-Theilen feline leukemia and sarcoma viruses: comparative studies of feline sarcoma virus with its natural helper virus and with Moloney murine sarcoma virus

Extrachromosomal DNA obtained from mink cells acutely infected with the Snyder-Theilen (ST) strain of feline sarcoma virus (feline leukemia virus) [FeSV(FeLV)] was fractionated electrophoretically, and samples enriched for FeLV and FeSV linear intermediates were digested with EcoRI and cloned in lambda phage. Hybrid phages were isolated containing either FeSV or FeLV DNA "inserts" and were characterized by restriction enzyme analysis, R-looping with purified 26 to 32S viral RNA, and heteroduplex formation. The recombinant phages (designated lambda FeSV and lambda FeLV) contain all of the genetic information represented in FeSV and FeLV RNA genomes but lack one extended terminally redundant sequence of 750 bases which appears once at each end of parental linear DNA intermediates. Restriction enzyme and heteroduplex analyses confirmed that sequences unique to FeSV (src sequences) are located at the center of the FeSV genome and are approximately 1.5 kilobase pairs in length. With respect to the 5'-3' orientation of genes in viral RNA, the order of genes in the FeSV genome is 5'-gag-src-env-c region-3'; only 0.9 kilobase pairs of gag and 0.6 kilobase pairs of env-derived FeLV sequences are represented in ST FeSV. Heteroduplex analyses between lambda FeSV or lambda FeLV DNA and Moloney murine sarcoma virus DNA (strain m1) were performed under conditions of reduced stringency to demonstrate limited regions of base pair homology. Two such regions were identified: the first occurs at the extreme 5' end of the leukemia and both sarcoma viral genomes, whereas the second corresponds to a 5' segment of leukemia virus "env" sequences conserved in both sarcoma viruses. The latter sequences are localized at the 3' end of FeSV src and at the 5' end of murine sarcoma virus src and could possibly correspond to regions of helper virus genomes that are required for retroviral transforming functions.     

Nucleotide sequences of feline sarcoma virus long terminal repeats and 5' leaders show extensive homology to those of other mammalian retroviruses.

The nucleotide sequences of the Gardner-Arnstein feline sarcoma virus (FeSV) long terminal repeat and the adjacent leader sequences 5' to the viral gag gene were determined. These were compared with homologous portions of Synder-Theilen FeSV and with previously published sequences for Moloney murine sarcoma virus and simian sarcoma virus proviral DNA. More than 75% of the residues in the FeSV R and U5 regions were homologous to sequences within the same regions of the other viral long terminal repeats. Unexpectedly, alignment of the FeSV sequences with those of the Moloney murine sarcoma and simian sarcoma viruses showed similar extents of homology within U3. The homologous U3 regions included the inverted repeats, a single set of putative enhancer sequences, corresponding to a "72-base-pair" repeat, and sequences, including the CAT and TATA boxes, characteristic of eucaryotic promotors. The 5' leader sequences of both FeSV strains included a binding site for prolyl tRNA and a putative splice donor sequence. In addition, the FeSV leader contained a long open reading frame which was adjacent to and in phase with the ATG codon at the 5' end of the FeSV gag gene. The open reading frame could code for a signal peptide of about 7.4 kilodaltons. Our results support the concept that the virogenic portions of both FeSV and simian sarcoma virus were ancestrally derived from viruses of rodent origin, with conservation of regulatory sequences as well as the viral structural genes.     

A new acute transforming feline retrovirus with fms homology specifies a C-terminally truncated version of the c-fms protein that is different from SM-feline sarcoma virus v-fms protein.

The HZ5-feline sarcoma virus (FeSV) is a new acute transforming feline retrovirus which was isolated from a multicentric fibrosarcoma of a domestic cat. The HZ5-FeSV transforms fibroblasts in vitro and is replication defective. A biologically active integrated HZ5-FeSV provirus was molecularly cloned from cellular DNA of HZ5-FeSV-infected FRE-3A rat cells. The HZ5-FeSV has oncogene homology with the fms sequences of the SM-FeSV. The genome organization of the 8.6-kilobase HZ5-FeSV provirus is 5' delta gag-fms-delta pol-delta env 3'. The HZ5-and SM-FeSVs display indistinguishable in vitro transformation characteristics, and the structures of the gag-fms transforming genes in the two viruses are very similar. In the HZ5-FeSV and the SM-FeSV, identical c-fms and feline leukemia virus p10 sequences form the 5' gag-fms junction. With regard to v-fms the two viruses are homologous up to 11 amino acids before the C terminus of the SM-FeSV v-fms protein. In HZ5-FeSV a segment of 362 nucleotides then follows before the 3' recombination site with feline leukemia virus pol. The new 3' v-fms sequence encodes 27 amino acids before reaching a TGA termination signal. The relationship of this sequence with the recently characterized human c-fms sequence has been examined. The 3' HZ5-FeSV v-fms sequence is homologous with 3' c-fms sequences. A frameshift mutation (11-base-pair deletion) was found in the C-terminal fms coding sequence of the HZ5-FeSV. As a result, the HZ5-FeSV v-fms protein is predicted to be a C-terminally truncated version of c-fms. This frameshift mutation may determine the oncogenic properties of v-fms in the HZ5-FeSV.     

Nature and distribution of feline sarcoma virus nucleotide sequences.

The genomes of three independent isolates of feline sarcoma virus (FeSV) were compared by molecular hybridization techniques. Using complementary DNAs prepared from two strains, SM- and ST-FeSV, common complementary DNA'S were selected by sequential hybridization to FeSV and feline leukemia virus RNAs. These DNAs were shown to be highly related among the three independent sarcoma virus isolates. FeSV-specific complementary DNAs were prepared by selection for hybridization by the homologous FeSV RNA and against hybridization by fline leukemia virus RNA. Sarcoma virus-specific sequences of SM-FeSV were shown to differ from those of either ST- or GA-FeSV strains, whereas ST-FeSV-specific DNA shared extensive sequence homology with GA-FeSV. By molecular hybridization, each set of FeSV-specific sequences was demonstrated to be present in normal cat cellular DNA in approximately one copy per haploid genome and was conserved throughout Felidae. In contrast, FeSV-common sequences were present in multiple DNA copies and were found only in Mediterranean cats. The present results are consistent with the concept that each FeSV strain has arisen by a mechanism involving recombination between feline leukemia virus and cat cellular DNA sequences, the latter represented within the cat genome in a manner analogous to that of a cellular gene.     

Transformation-defective mutants of feline sarcoma virus which express a product of the viral src gene.

Mink cell cultures infected with the Snyder-Theilen strain of feline sarcoma-leukemia virus were cloned from single cells under conditions favoring single virus-single cell interactions. The primary colonies included (i) typical feline sarcoma virus (FeSV)-transformed nonproducer clones, one of which segregated revertants, and (ii) FeSV-infected, phenotypically normal clones, three of which spontaneously converted to the transformed phenotype. The revertants and spontaneous transformants were compared with parental and sister clones expressing the opposite phenotype. Transformed subclones formed colonies in agar, were tumorigenic in nude mice, and failed to bind epidermal growth factor, whereas flat sister subclones were indistinguishable from uninfected mink cells in each of these assays. Sister subclones derived from the same infectious event contained FeSV proviruses integrated at the same molecular site, regardless of which phenotype was expressed. One revertant clone, however, lacked most FeSV proviral DNA sequences but retained terminal portions of the FeSV genome which persisted at the original site of proviral DNA insertion. Two flat subclones expressed viral RNA and the phosphorylated "gag-x" polyprotein (pp78gag-x) encoded by the gag and src sequences of the FeSV genome. Both of these clones were susceptible to retransformation by FeSV. Although unable to induce foci, the viruses rescued from these cells contained as much FeSV RNA as the focus-forming viruses rescued from transformed sister subclones and could be retransmitted to mink cells, again inducing FeSV gene products without signs of morphological transformation. We conclude that these FeSV genomes represent transformation-defective mutants.     

Structure of the feline c-fes/fps proto-oncogene: genesis of a retroviral oncogene.

The nucleotide sequence of the feline c-fes/fps proto-oncogene was analyzed. Comparison with v-fes and v-fps revealed that all v-fes/fps homologous sequences were dispersed over 11 kilobase pairs in 19 interspersed segments. All segments, numbered exon 1 to exon 19 as in the chicken and human loci, were flanked by consensus splice junctions. The putative promoter region contained a CATT sequence and three CCGCCC motifs which were also found in the human locus at similar positions. About 200 nucleotides downstream of a translational stop codon in exon 19, a putative poly(A) addition signal was identified. Using the putative translation initiation codon in exon 2, a 93,000-molecular-weight protein could be deduced. This protein resembled very well the putative protein of the human c-fes/fps proto-oncogene (94% overall homology) and, although less well, the putative protein of the chicken c-fes/fps proto-oncogene (70% overall homology). As far as the feline c-fes/fps proto-oncogene sequences transduced to the Gardner-Arnstein (GA) and Snyder-Theilen (ST) strains of feline sarcoma virus (FeSV) are concerned, homology in deduced amino acid sequences between the GA- and ST-v-fes viral oncogenes and the proto-oncogene was 99%. Analysis of the recombination junctions between feline leukemia virus and v-fes sequences in GA- and ST-FeSV proviral DNA revealed for the left-hand junction the involvement of homologous recombination, presumably at the DNA level. The right-hand junction, which appeared identical in the GA-FeSV and ST-FeSV genomes, could have been the result of a site-specific recombination at the RNA level.     

Nucleotide sequences of feline retroviral oncogenes (v-fes) provide evidence for a family of tyrosine-specific protein kinase genes

The nucleotide sequences encoding the transforming polyproteins of the Snyder-Theilen and Gardner-Arnstein strains of feline sarcoma virus (FeSV) have been determined. These sequences include a viral transforming gene (v-fes), derived from cellular proto-oncogene sequences (c-fes) of domestic cats by recombination with feline leukemia virus (FeLV). The v-fes sequences are predicted to encode a polypeptide domain strikingly similar to that specified by the transforming gene (v-fps) of the avian Fujinami sarcoma virus. In addition, the 3′ 0.8 kilobase pairs of v-fes encode amino acid sequences homologous to the carboxy-terminal portion of pp60^src, the transforming protein encoded by the avian Rous sarcoma virus src gene. Thus different feline and avian retroviral transforming genes, all of which encode functionally related proteins with associated tyrosine-specific kinase activities, must be derived from divergent members of the same protooncogene family.     

Monoclonal antibodies specific to transforming polyproteins encoded by independent isolates of feline sarcoma virus.

Hybridomas secreting monoclonal antibodies directed against polyprotein gene products of the Gardner, Snyder-Theilen, and McDonough strain of feline sarcoma virus have been isolated. Antibody produced by one hybridoma recognizes immunological determinants localized within a feline leukemia virus gag gene structural component (p15) common to polyproteins encoded by each feline sarcoma virus isolate while antibody produced by a second is specific for p30 determinants unique to P170gag-fms. Additional hybridomas secrete antibody directed against v-fes specific determinants common to the Gardner and Snyder-Theilen feline sarcoma virus-encoded polyproteins and to v-fms determinants unique to P170gas-fms polyprotein. GA P110gas-fes and ST P85gas-fes immunoprecipitated by antibody directed against p15 exhibit readily detectable levels of protein kinase activity but lack such activity when precipitated by antibody specific for their acquired sequence (v-fes) components. P170gas-fms immunoprecipitated by monoclonal antibody to either p15 or p30 lacks detectable levels of autophosphorylation but represents a substrate for the GA P110gag-fes and ST P85gag-fes enzymatic activities. These findings argue that the v-fes-associated protein kinase represents an intrinsic property of the v-fes gene product and recognizes tyrosine acceptor sites within polyprotein gene products of all three strains of feline sarcoma virus.     

The amino-terminal domain of the v-fms oncogene product includes a functional signal peptide that directs synthesis of a transforming glycoprotein in the absence of feline leukemia virus gag sequences.

The nucleotide sequence of a 5' segment of the human genomic c-fms proto-oncogene suggested that recombination between feline leukemia virus and feline c-fms sequences might have occurred in a region encoding the 5' untranslated portion of c-fms mRNA. The polyprotein precursor gP180gag-fms encoded by the McDonough strain of feline sarcoma virus was therefore predicted to contain 34 v-fms-coded amino acids derived from sequences of the c-fms gene that are not ordinarily translated from the proto-oncogene mRNA. The (gP180gag-fms) polyprotein was cotranslationally cleaved near the gag-fms junction to remove its gag gene-coded portion. Determination of the amino-terminal sequence of the resulting v-fms-coded glycoprotein, gp120v-fms, showed that the site of proteolysis corresponded to a predicted signal peptidase cleavage site within the c-fms gene product. Together, these analyses suggested that the linked gag sequences may not be necessary for expression of a biologically active v-fms gene product. The gag-fms sequences of feline sarcoma virus strain McDonough and the v-fms sequences alone were inserted into a murine retroviral vector containing a neomycin resistance gene. Both constructs were biologically active when transfected into NIH 3T3 cells and produced morphologically transformed foci at equivalent efficiencies. When transfected into a cell line (psi 2) expressing complementary viral gene functions, G418-resistant (Neor) cells containing either of these vector DNAs produced high titers of transforming viruses. Analysis of proteins produced in cells containing the vector lacking gag gene sequences showed that gP180gag-fms was not synthesized, whereas normal levels of both immature gp120v-fms and mature gp140v-fms were detected. The glycoprotein was efficiently transported to the cell surface, and it retained wild-type tyrosine kinase activity. We conclude that a cryptic hydrophobic signal peptide sequence in v-fms was unmasked by gag deletion, thereby allowing the correct orientation and transport of the v-fms gene product within membranous organelles. It seems likely that the proteolytic cleavage of gP180gag-fms is mediated by signal peptidase and that the amino termini of gp140v-fms and the c-fms gene product are identical.     

Translational products encoded by newly acquired sequences of independently derived feline sarcoma virus isolates are structurally related

Polyproteins encoded by several independent isolates of feline sarcoma virus (FeSV) were analyzed with respect to molecular weight, extent of phosphorylation, and tryptic peptide composition. As previously reported, cells nonproductively transformed by the Gardner strain of FeSV express a polyprotein which has a molecular weight of approximately 115,000 and contains feline leukemia virus p15, p12, and minor portion of p30. In addition, a major 72,000-dalton possible cleavage product can be identified. Snyder-Theilen FeSV-transformed cells express a major polyprotein of approximately 115,000 daltons and a second highly related 80,000-dalton protein. The p12 structural component of Gardner FeSV P115, but not Snyder-Theilen FeSV 115, corresponds to feline leukemia virus subgroup A with respect to immunological type specificity, a finding consistent with the independent origin of these viruses. Tryptic peptide analysis revealed five methionine-containing peptides specific to the nonstructural portion of Gardner FeSV 115, three of which were also represented in Snyder-Theilen FeSV P115, three of which were also represented in Snyder-Theilen FeSV P115. None of these [35S]methionine-labeled tryptic peptides were present in translational products representative of the complete feline leukemia virus subgroup A genome, including Pr180gag-pol, Pr65gag, and Pr82env. Similarly phosphorylated tryptic peptides within the structural (p12) and nonstructural components of Gardner FeSV P115 and Snyder-Theilen FeSV P115 Are highly related. These findings support the possibility that acquired sequences of two independently derived isolates of FeSV encode structurally related proteins.     

Three independent isolates of feline sarcoma virus code for three distinct gag-x polyproteins.

Cells nonproductively transformed by the Snyder-Theilen, Gardner-Arnstein, and McDonough strains of feline sarcoma virus synthesize gag-x polyproteins of 78,000, 100,000, and 180,000 daltons, respectively. These feline sarcoma virus-coded products were precipitated by antisera to polypeptides encoded by the gag gene of feline leukemia virus and by rat antisera raised to feline sarcoma virus-transformed rat tumor cells. Precipitation with rat antisera absorbed with feline leukemia virus showed that the x-portions of the three gag-x proteins were each antigenically distinct, suggesting that the src genes of the three independent isolates are not identical. Anti-x sera did not precipitate products from radiolabeled cat lymphoid tumor cells (FL74) and therefore lacked reactivity to the feline leukemia virus-induced tumor-specific antigen, FOCMA.     

Gene products of McDonough feline sarcoma virus have an in vitro-associated protein kinase that phosphorylates tyrosine residues: lack of detection of this enzymatic activity in vivo.

The primary translational product of the McDonough (SM) strain of feline sarcoma virus (FeSV) is a 180,000-dalton molecule, SM P180, that contains the p15-p12-p30 region of the FeLV gag gene-coded precursor protein and a sarcoma virus-specific polypeptide. In addition, cells transformed by SM-FeSV express a 120,000-dalton molecule, SM P120, that is highly related to the non-helper virus domain of SM P180. Both SM-FeSV gene products were found to be intimately associated with the membrane fraction of SM-FeSV-transformed cells. Immunoprecipitates containing SM P180 and SM P120 exhibited a protein kinase activity capable of phosphorylating tyrosine residues of both viral gene products but not immune immunoglobulin G molecules. By independently immunoprecipitating each of the two SM-FeSV proteins we found that most of the tyrosine-specific phosphorylating activity was associated with the SM P120 molecule. In vivo analysis of 32P-labeled SM P180 and SM P120 revealed their phosphoprotein nature; however, both molecules exhibited low levels of phosphorylation and did not contain phosphotyrosine residues. Finally, we did not detect any significant elevation in the levels of phosphotyrosine in the protein fraction of SM-FeSV transformants. Thus, if SM-FeSV were to induce malignant transformation by a mechanism involving phosphorylation of tyrosine residues, the viral gene products must interact with a small subset of cellular proteins that do not represent a significant fraction of the total cellular protein content.     

The Parodi-Irgens feline sarcoma virus and simian sarcoma virus have homologous oncogenes, but in different contexts of the viral genomes

We have identified the oncogene and the putative transforming protein of the Parodi-Irgens feline sarcoma virus (PI-FeSV). The PI-FeSV is defective and needs a helper virus for its replication. The v-onc sequences in the PI-FeSV were found to be related to the v-sis sequences of the simian sarcoma virus (SSV). PI-FeSV nonproducer cells express two viral RNAs, a 6.8-and a 3.3-kilobase RNA. The 6.8-kilobase RNA contains gag, sis, and env sequences but lacks the pol gene. The 3.3-kilobase RNA, on the other hand, contains only env sequences. We have detected one feline leukemia virus-related protein product in these cells, namely, a 76-kilodalton protein which contains determinants of the feline leukemia virus gag proteins p15 and p30. The v-sis sequences in the PI-FeSV have been located near the 5' end of the viral genome. Taken together, these results imply that the p76 protein contains both feline leukemia virus gag and sis sequences and probably is the transforming protein of this virus. In contrast, in SSV the sis sequences are located towards the 3' end of the viral genome, and the sis protein is thought to be expressed via a subgenomic RNA. PI-FeSV and SSV therefore use different schemes to express their onc-related sequences. The v-sis sequences in the PI-FeSV contain restriction sites which reflect the different origin of the v-sis sequences in the PI-FeSV and SSV. The homologous oncogenes of the PI-FeSV and SSV thus were transduced by two different retroviruses, feline leukemia virus and the simian sarcoma-associated virus, apparently from the genomes of different species.     

Biochemical and immunological characterization of polyproteins coded for by the McDonough, Gardner-Arnstein, and Snyder-Theilen strains of feline sarcoma virus.

The McDonough (SM), Gardner-Arnstein (GA), and Snyder-Theilen (ST) strains of feline sarcoma virus (FeSV) code for high-molecular-weight polyproteins that contain varying amounts of the amino-terminal region of the FeLV gag gene-coded precursor protein and a polypeptide(s) of an as yet undetermined nature. The SM-FeSV primary translational product is a 180,000-dalton polyprotein which is immediately processed into a highly unstable 60,000-dalton molecule containing the p15-p12-p30 fragment of the FeLV gag gene-coded precursor protein and a 120,000-dalton FeSV-specific polypeptide. The GA- and ST-FeSV genomes code for polyproteins of 95,000 and 85,000 daltons, respectively, which in addition to the amino-terminal moiety (p15-12 and a portion of p30) of the FeLV gag gene-coded precursor protein also contain FeSV-specific polypeptides. However, the GA- and ST-FeSV polyproteins appear to be relatively stable molecules (half-lives of around 16 h) and are not significantly processed into smaller polypeptides. Immunological and biochemical analysis of each of the above FeSV translational products revealed that the sarcoma-specific regions of the GA- and ST-FeSV polyproteins are antigenically cross-reactive and exhibit common methionine-containing peptides. These findings favor the concept that these sarcoma-specific polypeptides are coded for by the similar subsets of cellular sequences incorporated into the GA- and ST-FeSV genomes during the generation of these transforming agents.     

Cellular transformation by subgenomic feline sarcoma virus DNA

The genome of the Snyder-Theilen strain of feline sarcoma virus (ST-FeSV) is a 4.3-kilobase-pair (kbp) RNA molecule that contains a 1.5-kbp cellular insertion (fes gene) flanked by feline leukemia virus sequences at its 5' end (1.6 kbp) and 3' end (1.2 kbp) (Sherr et al., J. Virol. 34:200-212, 1980). DNA transfection techniques have been utilized to determine the regions of the ST-FeSV genome involved in malignant transformation. I have found that the 3.7-kbp 5'-end fragment of the ST-FeSV provirus (which corresponds to the 3.4-kbp 5'-end fragment of the viral genome) is sufficient to transform NIH/3T3 fibroblasts. Enzymes that cleave the ST-FeSV provirus DNA within the feline leukemia virus gag gene sequences or within the fes gene abolished the transforming activity. Preservation of the proviral large terminal repeats was also required for transformation. Transformed NIH/3T3 cells obtained by transfection of total or subgenomic ST-FeSV DNA expressed normal levels of the ST-FeSV gene product ST P85 and of its associated protein kinase activity. Furthermore, these cells contained high levels of phosphotyrosine residues, a biochemical marker associated with cellular transformation induced by certain retroviruses including ST-FeSV. These results, taken together, strongly support the concept that only those ST-FeSV proviral sequences necessary for ST P85 expression are involved in malignant transformation.     

Preparation of rat monoclonal antibodies to epitopes encoded by the viral oncogene (v-fms) of McDonough feline sarcoma virus

The McDonough strain of feline sarcoma virus (SM-FeSV) contains a viral oncogene, v-fms, transduced from cat cellular genetic sequences designated c-fms. Monoclonal antibodies reactive to antigenic determinants encoded by v-fms were prepared by immunizing rats with live, syngeneic SM-FeSV-transformed cells, and fusing splenic lymphocytes from a tumor-bearing animal with cultured rat myeloma cells. Culture supernatants from hybrids producing antibodies to epitopes encoded by v-fms were identified by immunoprecipitation of radiolabeled polypeptides from SM-FeSV-transformed mink cells. Four positive hybrids were cloned twice in soft agar, established as stable lines, and grown in defined serum-free medium to facilitate purification of homogeneous antibodies. The monoclonal antibodies were used to assay SM-FeSV-specific products by "immunoblotting" of electrophoretically separated proteins, and by fixed-cell immunofluorescence.     

Isolation and characterization of a human locus homologous to the transforming gene (v-fes) of feline sarcoma virus

A single locus (designated c-fes) in the human genome which exhibits homology to the transformation-specific onc gene (v-fes) of Snyder-Theilen feline sarcoma virus was identified by the Southern blot technique. Recombinant clones containing 16- to 18-kilobase inserts of human DNA including the c-fes locus were constructed. Restriction endonuclease mapping of these clones verified their identity with native human c-fes and demonstrated the presence of at least two sequences in human c-fes interrupting v-fes-homologous regions. The v-fes-homologous locus in the human genome spans about 4 kilobases. The 5'-3' orientation of the c-fes clones with respect to feline sarcoma virus proviral DNA was determined. The region of the human genome that is homologous to v-fes is proximal to the highly reiterated human Alu sequence but not to the highly reiterated human alphoid sequence.