Human papillomavirus type 16 (HPV-16) genomes integrated in head and neck cancers and in HPV-16-immortalized human keratinocyte clones express chimeric virus-cell mRNAs similar to those found in cervical cancers

Many human papillomavirus (HPV)-positive high-grade lesions and cancers of the uterine cervix harbor integrated HPV genomes expressing the E6 and E7 oncogenes from chimeric virus-cell mRNAs, but less is known about HPV integration in head and neck cancer (HNC). Here we compared viral DNA status and E6-E7 mRNA sequences in HPV-16-positive HNC tumors to those in independent human keratinocyte cell clones derived from primary tonsillar or foreskin epithelia immortalized with HPV-16 genomes. Three of nine HNC tumors and epithelial clones containing unintegrated HPV-16 genomes expressed mRNAs spliced from HPV-16 SD880 to SA3358 and terminating at the viral early gene p(A) signal. In contrast, most integrated HPV genomes in six HNCs and a set of 31 keratinocyte clones expressed HPV-16 major early promoter (MEP)-initiated mRNAs spliced from viral SD880 directly to diverse cellular sequences, with a minority spliced to SA3358 followed by a cellular DNA junction. Sequence analysis of chimeric virus-cell mRNAs from HNC tumors and keratinocyte clones identified viral integration sites in a variety of chromosomes, with some located in or near growth control genes, including the c-myc protooncogene and the gene encoding FAP-1 phosphatase. Taken together, these findings support the hypothesis that HPV integration in cancers is a stochastic process resulting in clonal selection of aggressively expanding cells with altered gene expression of integrated HPV genomes and potential perturbations of cellular genes at or near viral integration sites. Furthermore, our results demonstrate that this selection also takes place and can be studied in primary human keratinocytes in culture.     

Characterization of flat revertant cells isolated from simian virus 40-transformed mouse and rat cells which contain multiple copies of viral genomes.

Eight clones of flat revertants were isolated by negative selection from simian virus 40 (SV40)-transformed mouse and rat cell lines in which two and six viral genome equivalents per cell were integrated, respectively. These revertants showed either a normal cell phenotype or a phenotype intermediate between normal and transformed cells as to cellular morphology and saturation density and were unable to grow in soft agar medium. One revertant derived from SV40-transformed mouse cells was T antigen positive, whereas the other seven revertants were T antigen negative. SV40 could be rescued only from the T-antigen-positive revertant by fusion with permissive monkey cells. The susceptibility of the revertants to retransformation by wild-type SV40 was variable among these revertants. T-antigen-negative revertants from SV40-transformed mouse cells were retransformed at a frequency of 3 to 10 times higher than their grandparental untransformed cells. In contrast, T-antigen-negative revertants from SV40-transformed rat cells could not be retransformed. The arrangement of viral genomes was analyzed by digestion of cellular DNA with restriction enzymes of different specificity, followed by detection of DNA fragments containing a viral sequence and rat cells were serially arranged within the length of about 30 kilobases, with at least two intervening cellular sequences. A head-to-tail tandem array of unit length viral genomes was present in at least one insertion site in the transformed rat cells. All of the revertants had undergone a deletion(s), and only a part of the viral genome was retained in T-antigen-negative revertants. A relatively high frequency of reversion in the transformed rat cells suggests that reversion occurs by homologous recombination between the integrated viral genomes.     

Recombination between a defective retrovirus and homologous sequences in host DNA: reversion by patch repair.

The genomes of mammalian species contain multiple copies of sequences homologous to exogenous retroviruses. When a mutant retrovirus carrying a lethal deletion in an essential viral gene was introduced into mammalian cells, revertant viruses appeared and spread throughout the culture. Analysis of one such revertant showed that the mutation had been repaired by homologous recombination with endogenous sequences. Our results suggest that defective retroviruses can draw upon the genetic complement of the host cell to repair lesions in viral genes.     

Genetic transformation of somatic cells. VI. Transfer of the trait of the rate of loss of transformant phenotype by means of DNA from cells containing the thymidine kinase gene of the herpes simplex virus

Using dot-hybridization with thymidine kinase gene (tk gene) of Herpes simplex virus type 1 (HSV 1) of DNA preparations obtained from isolated metaphase chromosomes and lysate fractions of metaphase cells, which presumably contain smaller particles compared to metaphase chromosomes, it has been shown that the tk gene of HSV 1 is localized in chromosomes of cells of transformant clones unstable in TK+-phenotype. The DNA isolated from the metaphase chromosomes from cells of transformant clones is 1.5- or 2-fold more efficient in transforming TK-Chinese hamster cells than is the total high molecular weight DNA from the same cells. Upon transformation of TK- cells by the high molecular weight DNA from the tk gene of HSV 1-containing clones, varying in the rate of the loss of TK+-phenotypes, the character "rate of the loss of transformant phenotype" is transferred together with the tk gene of HSV 1 in 22% of cases. Cells of rerevertant clones, produced from TK- subclones of transformant clones, display the rate of the loss of transformant phenotype characteristic of cells of parental TK+-clones. A comparison of the results allows a conclusion that DNA sequences, determining the character "rate of the loss of transformant phenotype", are linked tightly with the transforming DNA proper containing the tk gene of HSV 1, but are not localized inside such a DNA.     

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.     

Organization and primary sequence of multiple genes coding for the apopolysialoglycoproteins of rainbow trout

We have shown that the mRNAs for apopolysialoglycoproteins (apoPSGP) of rainbow trout contain various numbers of a repetitive sequence of 39 base-pairs encoding mature apoPSGP, and that this sequence is bordered by highly homologous 5' and 3' regions encoding pre-, pro- and telopeptides. These mRNAs are thought to be transcribed from different genes that constitute a large multiple gene family (more than 100 members). Here, we have determined the structures of several members of the apoPSGP gene family. The results show that two of three genomic DNA fragments contain two independent apoPSGP genes in the same orientation with unrelated sequences intervening. Five characterized genes have essentially the same organization and sequence. Each gene has four exons, and CAAT and TATA sequences were found in the 5'-flanking regions. However, two noteworthy differences were observed among the five genes; a diversity in the number of the 39 base-pair repeats, also observed among the cDNA clones, and a one-base polymorphism in the 39 base-pair repeat, which causes an amino acid change. This polymorphism was not detected among the cDNA clones obtained. The boundary positions of the genes are various and contain no transposon-like structures. The variation in the number of repeats and the absence of a rule for bordering positions of the genes suggest that apoPSGP genes may have been amplified by gene duplications, unequal recombination, and selection of chromosomes having larger numbers of apoPSGP genes.     

Structural relationship between a normal chicken DNA locus and the transforming gene of the avian acute leukemia virus MC29.

We screened a recombinant chicken DNA/lambda phage library for sequences homologous to the transformation-specific sequences of the avian acute leukemia virus MC29 by hybridization with molecularly cloned MC29 proviral DNA. Three cellular DNA clones were found and compared with each other and with the viral genome by physical mapping with restriction endonucleases and by heteroduplex analysis. These experiments indicated that the three cellular clones overlap and represent a single cellular locus. The RNA genome of MC29 and normal cell DNA share a homologous region of 1.6 kilobases which is interrupted in the cellular DNA by 1.0 kilobase of sequences not present in the viral genome. Hybridization of the cloned cellular DNA to viral RNA and analysis of the protected viral RNA by fingerprinting techniques indicated that there is extensive sequence homology between the helper virus-unrelated mcv sequences of the viral RNA and the cellular DNA, with only minor base differences. The cellular mcv locus, however, lacks all helper virus-related sequences of MC29, including those of the partial viral gag gene which, together with mcv, encodes the probable transforming protein of MC29. We conclude that although the mcv locus of the normal cell does not represent a complete structural homolog to the onc gene of MC29, it is probably the precursor to the onc-specific sequence in the virus.     

Localization of the human alpha-globin structural gene to chromosome 16 in somatic cell hybrids by molecular hybridization assay

We have used 16 human × mouse somatic cell hybrids containing a variable number of human chromosomes to demonstrate that the human α-globin gene is on chromosome 16. Globin gene sequences were detected by annealing purified human α-globin complementary DNA to DNA extracted from hybrid cells. Human and mouse chromosomes were distinguished by Hoechst fluorescent centromeric banding, and the individual human chromosomes were identified in the same spreads by Giemsa trypsin banding. Isozyme markers for 17 different human chromosomes were also tested in the 16 clones which have been characterized. The absence of chromosomal translocation in all hybrid clones strongly positive for the α-globin gene was established by differential staining of mouse and human chromosomes with Giemsa 11 staining. The presence of human chromosomes in hybrid cell clones which were devoid of human α-globin genes served to exclude all human chromosomes except 6, 9, 14 and 16. Among the clones negative for human α-globin sequences, one contained chromosome 2 (JFA 14a 5), three contained chromosome 4 (AHA 16E, AHA 3D and WAV R4D) and two contained chromosome 5 (AHA 16E and JFA14a 13 5) in >10% of metaphase spreads. These data excluded human chromosomes 2, 4 and 5 which had been suggested by other investigators to contain human globin genes. Only chromosome 16 was present in each one of the three hybrid cell clones found to be strongly positive for the human α-globin gene. Two clones (WAIV A and WAV) positive for the human α-globin gene and chromosome 16 were counter-selected in medium which kills cells retaining chromosome 16. In each case, the resulting hybrid populations lacked both human chromosome 16 and the α-globin gene. These studies establish the localization of the human α-globin gene to chromosome 16 and represent the first assignment of a nonexpressed unique gene by direct detection of its DNA sequences in somatic cell hybrids.     

Cytogenetic localization of 44 new coding sequences in the horse

The purpose of this study was to increase the number of genes assigned by in situ hybridization to equine chromosomes and thus the number of links for comparative mapping with other species. Forty-four new sequences were added to the horse cytogenetic map by FISH mapping of BAC clones containing genes (35) or ESTs (9). Three approaches were developed: use of horse BAC clones screened with (i) horse EST primers, (ii) interspecific consensus intraexonic primers, and (iii) use of goat BAC containing genes previously localized on goat chromosomes. Present data suggest that the second approach is the most promising. A total of 46 segments containing one or several genes could be compared, among which 40 loci could be included in 16 synteny groups between human and horse, displaying one ordered segment and several breaking points along chromosomes. All single BAC localizations confirm the most recent mapping data. Twenty-six out of 31 chromosomes now contain a gene mapped by in situ hybridization, and 14 new arm-to-arm segment homologies were revealed. Received: 2 May 2000 / Accepted: 27 July 2000     

Structural analysis of a Lotus japonicus genome. II. Sequence features and mapping of sixty-five TAC clones which cover the 6.5-mb regions of the genome.

Sixty-five TAC (transformation-competent artificial chromosomes) clones were selected from a genomic library of Lotus japonicus accession MG-20 based on the sequence information of expressed sequences tags (ESTs), cDNA and gene information, and their nucleotide sequences were determined. The average insert size of the TAC clone was approximately 100 kb, and the total length of the sequenced regions in this study is 6,556,100 bp. Together with the nucleotide sequences of 56 TAC clones previously reported, the regions sequenced so far total 12,029,295 bp. By comparison with the sequences in protein and EST databases and by analysis with computer programs for gene modeling, a total of 711 potential protein-encoding genes with known or predicted functions, 239 gene segments and 90 pseudogenes were identified in the newly sequenced regions. The average gene density assigned so far was 1 gene/9140 bp. The average length of the assigned genes was 2.6 kb, which is considerably larger than that assigned in the Arabidopsis thaliana genome (1.9 kb for 6451 genes). Introns were identified in approximately 73% of the potential genes, and the average number and length of the introns per gene were 3.4 and 377 bp, respectively. Simple sequence repeat length polymorphism (SSLP) or derived cleaved amplified polymorphic sequence (dCAPS) markers were generated based on the nucleotide sequences of the genomic clones obtained, and each clone was mapped onto the linkage map using the F2 mapping population derived from a cross of two accessions of L. japonicus, Gifu B-129 and Miyakojima MG-20. The sequence data, gene information and mapping information are available through the World Wide Web at http://www.kazusa.or.jp/lotus/.     

Gene expression modulation is associated with gene amplification, supernumerary chromosomes and chromosome loss in antimony-resistant Leishmania infantum

Antimonials remain the first line drug against the protozoan parasite Leishmania but their efficacy is threatened by resistance. We carried out a RNA expression profiling analysis comparing an antimony-sensitive and -resistant (Sb2000.1) strain of Leishmania infantum using whole-genome 70-mer oligonucleotide microarrays. Several genes were differentially expressed between the two strains, several of which were found to be physically linked in the genome. MRPA, an ATP-binding cassette (ABC) gene known to be involved in antimony resistance, was overexpressed in the antimony-resistant mutant along with three other tandemly linked genes on chromosome 23. This four gene locus was flanked by 1.4 kb repeated sequences from which an extrachromosomal circular amplicon was generated in the resistant cells. Interestingly, gene expression modulation of entire chromosomes occurred in the antimony-resistant mutant. Southern blots analyses and comparative genomic hybridizations revealed that this was either due to the presence of supernumerary chromosomes or to the loss of one chromosome. Leishmania parasites with haploid chromosomes were viable. Changes in copy number for some of these chromosomes were confirmed in another antimony-resistant strain. Selection of a partial revertant line correlated antimomy resistance levels and the copy number of aneuploid chromosomes, suggesting a putative link between aneuploidy and drug resistance in Leishmania.     

Characterization and chromosome assignment of the human homolog of int-2, a potential proto-oncogene.

int-2 is one of two cellular genes (int-1 and int-2) currently implicated in the genesis of mammary carcinomas by mouse mammary tumor virus and may constitute a novel cellular proto-oncogene. Using low-stringency hybridization with mouse int-2 probes, we established that homologous genes exist in a variety of mammalian species, including humans, but failed to detect related sequences in other classes and phyla. Recombinant bacteriophage clones and a single cosmid encompassing the human int-2 gene were isolated and characterized by restriction enzyme mapping. A survey of nine primary human breast tumors, three breast tumor cell lines, and three normal individuals revealed no evidence for gross amplification or rearrangement of the int-2 locus. Three distinct restriction fragment length polymorphisms were observed which could prove useful in future linkage studies. By a combination of in situ hybridization of metaphase chromosomes and somatic cell genetics, the human int-2 gene was mapped to chromosome 11, band q13.     

Production of avian oncoviral subgroups after multiple infection.

The number of different oncoviral env genes that can be expressed by a single chicken embryo fibroblast was investigated. Fibroblasts were infected with one to three subgroups of Rous-associated virus, which is a nontransforming avian oncovirus, then superinfected with a transforming virus, Rous sarcoma virus, of a different subgroup. The subgroups of viruses released by the resulting clones were analyzed. When two viral subgroups were used for preinfection, all the resulting clones produced transforming virus particles having the subgroup of the superinfecting virus, and most clones produced transforming virus particles of all the infecting viral subgroups. However, when cells were preinfected with three viral subgroups, many of the resulting clones did not produce transforming virus particles having the subgroup of the superinfecting virus, and only 1 of 23 clones produced transforming particles of all the infecting viral subgroups. DNA annealing experiments showed that cells infected with three or four viral subgroups had an additional 8 to 20 copies of proviral DNA per cell. Finally, most clones resulting from cells simultaneously infected with three or four viral subgroups were able to produce virus of all infecting subgroups. It appears that the number of exogenous oncoviral env genes that can be expressed by a single cell is limited, and in the range of 4 to 8-20 per cell.     

Characterization of Morphologic Revertants of Murine and Avian Sarcoma Virus-Transformed Cells

Morphologic revertants which contain avian or murine sarcoma viruses have previously been isolated at low frequency from clonal lines of transformed mammalian cells. In the present study, these lines have been further characterized. They are indistinguishable from nontransformed parent cell lines with respect to parameters such as saturation density and colony formation in depleted medium or on monolayers of contact-inhibited cells. The rate of glucose uptake had also reverted to normal. The malignant potential of one of the revertant lines was examined and found to be markedly reduced compared to that of the corresponding transformed cells. The differences in the susceptibilities of revertant cells to retransformation by the same or other oncogenic viruses suggest that different cellular genes may be involved in expression of transformation by various tumor viruses.     

Analysis of integrated human papillomavirus type 16 DNA in cervical cancers: amplification of viral sequences together with cellular flanking sequences.

We have isolated four clones of integrated human papillomavirus type 16 (HPV-16) DNA from four different primary cervical cancer specimens. All clones were found to be monomeric or dimeric forms of HPV-16 DNA with cellular flanking sequences at both ends. Analysis of the viral sequences in these clones showed that E6/E7 open reading frames and the long control region were conserved and that no region specific for the integration was detected. Analysis of the cellular flanking sequences revealed no significant homology with any known human DNA sequences, except Alu sequences, and no homology among the clones, indicating no cellular sequence specific for the integration. By probing with single-copy cellular flanking sequences from the clones, it was demonstrated that the integrated HPV-16 DNAs, with different sizes in the same specimens, shared the same cellular flanking sequences at the ends. Furthermore, it was shown that the viral sequences together with cellular flanking sequences were amplified. The possible process of viral integration into cell chromosomes in cervical cancer is discussed.     

CpG island libraries from human Chromosomes 18 and 22: landmarks for novel genes

CpG islands are found at the 5′ end of approximately 60% of human genes and so are important genomic landmarks. They are concentrated in early-replicating, highly acetylated gene-rich regions. With respect to CpG island content, human Chrs 18 and 22 are very different from each other: Chr 18 appears to be CpG island poor, whereas Chr 22 appears to be CpG island rich. We have constructed and validated CpG island libraries from flow-sorted Chrs 18 and 22 and used these to estimate the difference in number of CpG islands found on these two chromosomes. These libraries contain normalized collections of sequences from the 5′ end of genes. Clones from the libraries were sequenced and compared with the sequence databases; one third matched ESTs, thus anchoring these ESTs at the 5′ end of their gene. However, it was striking that many clones either had no match or matched only existing CpG island clones. This suggests that a significant proportion of 5′ gene sequences are absent from databases, presumably either because they are difficult to clone or the gene is poorly expressed and/or has a restricted expression pattern. This point should be taken into consideration if the currently available libraries are those used for the elucidation of complete, as opposed to partial, gene sequences. The Chr 18 and 22 CpG island libraries are a sequence resource for the isolation of such 5′ gene sequences from specific human chromosomes. Received: 15 November 1999 / Accepted: 31 January 2000     

Assignment of 118 novel cDNAs of cynomolgus monkey brain to human chromosomes

In order to isolate genes that may not be represented in current human brain cDNA libraries, we have sequenced about 20,000 sequence tags of cDNA clones derived from cerebellum and parietal lobe of cynomolgus monkeys (Macaca fascicularis). We determined the entire cDNA sequence of approximately 700 clones whose 5'-terminal sequences showed no homology to annotated putative genes or expressed sequence tags in current databases of genetic information. From this, 118 clones with sequences encoding novel open reading frames of more than 100 amino acid residues were selected for further analysis. To localize the genes corresponding to these 118 newly identified cDNA clones on human chromosomes, we performed a homology search using the human genome sequence and fluorescent in situ hybridization. In total, 108 of 118 clones were successfully assigned to specific regions of human chromosomes. This result demonstrates that genes expressed in cynomolgus monkey are highly conserved throughout primate evolution, and that virtually all had human homologs. Furthermore, we will be able to discover novel human genes in the human genome using monkey homologs as probes.