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Evolutionarily new sequences expressed in tumors

Evolutionarily new sequences expressed in tumors Background: Earlier we suggested the concept of the positive evolutionary role of tumors. According to this concept, tumors provide conditions for the expression of evolutionarily new and/ or sleeping genes in their cells. Thus, tumors are considered as evolutionary proving ground or reservoir of expression. To support this concept we have previously characterized in silico and experimentally a new class of human tumor-related transcribed sequences. Results: In this article we describe results of further studies of previously described tumor-related sequences. The results of molecular phylogeny studies, Southern hybridization experiments and computational comparison with genomes of other species are presented. Conclusion: These results suggest that these previously described tumor-related human transcripts are also relatively evolutionarily new. Background i.e., the tumor-specificity of expression of these sequences In previous studies [1,2], we formulated the concept of [4]. the positive evolutionary role of tumors. According to this concept, tumors provide conditions for the expression of To experimentally examine our prediction [1,2] that at evolutionarily new and/or sleeping genes in their cells. least some tumor-related sequences are evolutionarily Thus, tumors are considered as an evolutionary proving new, we performed Southern hybridization of our newly ground of expression. described tumor-related sequences with genomic DNA from different animal species. Hybridization was found In earlier work using the computational differential dis- only with human and orangutan DNA, with one excep- play approach, we identified a considerable number of tion in which a signal was also developed with chicken human tumor-related expressed sequence tag (EST) clus- DNA. ters many of which had not been described previously [3]. Experimental data confirmed the results obtained in silico, We performed a search for ortholog sequences in fugu, tetraodon, zebrafish, frog, chicken, rat, mouse, cow, dog, Page 1 of 10 (page number not for citation purposes) Infectious Agents and Cancer 2006, 1:8 http://www.biomedcentral.com/1750-9378/1/8 macaque, and chimpanzee genomes using cross-species Bank:AL040372], now in cluster Hs.133294; chained alignments. This search confirmed that our newly [GenBank:AI952931] from cluster Hs.128594 (former ] from cluster described tumor-related transcripts are relatively evolu- Hs.67624); and [GenBank:AI792557 tionarily new, with some of their orthologs having origi- Hs.133107. nated in mammals and others in primates. PCR analysis and Southern hybridization PCR experiments with specific primers were performed on We performed Southern hybridization of [α- P]-labeled a panel of DNAs from different primates. Amplified frag- sequence-specific fragments with genomic DNA from ments were cloned and sequenced, and their molecular eleven different animal species: lamprey, fish, frog, phylogeny was studied. The results show that these chicken, pigeon, mouse, rat, guinea pig, sheep, horse, and sequences form well-defined phylogenetic clusters which human. Southern hybridization analysis reveals only correspond to the phylogeny of primates as previously homology sequences in chicken genome for AA166653- understood. specific probe. In addition, we were able to demonstrate by Southern hybridization that a sequence homologous Taken together, our Southern hybridization, molecular to the human AA166653-specific 11.2-kb fragment is phylogeny, and comparative genomics data support our present in orangutan DNA [see Additional file 1]. prediction [1,2] that evolutionarily new and/or sleeping sequences may be specifically expressed in tumor cells. Therefore, we performed PCR amplification of sequence- specific fragments on the panel of primate DNAs. The results of PCR experiments and comparative genomics Results Transcribed sequences analyzed data obtained by homology analysis of these tumor- Because of the constant rebuilding of the UniGene clus- related sequences within primate DNA are presented in ters and EST shuffling between them, we cannot follow Table 1. As follows from the results shown in Table 1, the history of each cluster. Clusters very often do not mark sequences homologous to tumor-related human EST are a specific transcript, but a set of transcripts whose genome found in a variety of primates. mapping regions are often neighboring but may be not overlapping. Therefore, we selected ESTs which were used AL040372- and AI792557-specific tumor-related human for primer design in our previous investigations [3,4] and sequences are found in the majority of primate species followed the history of their sequences in UniGene. studied, even in the most archaic. The AI792557-homolo- gous sequence is not found in lemurs and colobus mon- In this paper, we present analyses of the following ESTs keys. The AA166653-homologous sequence is present (UniGene buid 185): [GenBank:AA166653], now in clus- only in apes and macaques. The AI952931-homologous ter Hs.426704 (former Hs. 154173); [Gen- sequence is found in apes, new world monkeys, and Table 1: Results of PCR experiments* and comparative genomics analysis within primates. Superfamily Species/Transcript (EST Clusters) #1 #2 #3 #4 Platyrrhini Lemur catta + --- New World monkeys Ateles fusciceps + - + + Callimico goeldii + - + + Cercopitecoidea Colobus guereza + --- Old World monkeys Erythrocebus patas + - - + Cercopithecus aethiops + - - + Macaca mulatta (+)** (+) (+) (+) Hominoidea Hylobates concolor + - + + Apes and Human Pongo pygmaeus (sumatran) + + + + Pongo pygmaeus (bornean) -- + + Gorilla gorilla (sample 1) + + + + Gorilla gorilla (sample 2) -- +/-*** + Pan troglodytes (sample 1) + + + (+) Pan troglodytes (sample 2) + + + (+) Homo sapiens + + + + * DNA samples were ranged according to the existing classification of primates [5]. ** Data in brackets are results of comparative genomics analysis. *** Weak signal. #1-AL040372, Hs.133294; #2-AA166653, Hs.426704; #3-AI952931, Hs.128594; #4-AI792557, Hs.133107 Page 2 of 10 (page number not for citation purposes) Infectious Agents and Cancer 2006, 1:8 http://www.biomedcentral.com/1750-9378/1/8 macaques. No sequences discussed in this article could be complete repeating unit. For this cluster, we used PCR amplified by PCR on a DNA evolutionary panel with primers specific to sequences located on chromosome 2. genomic DNA from species non-primate species using the According to our experimental data [4], this sequence is selected primers. expressed in carcinomas only. This sequence has not been found in any sequenced mammalian genome except rhe- Comparative genomics and bioinformatics analyses sus and chimpanzee, with similarity levels of 88% and Hs.133107 cluster consists of spliced mRNAs, but the 99%, respectively ([see Additional file 2], Table 2). In the studied sequence AI792557 is a short, unspliced EST and chimpanzee genome, the Hs.426704 locus underwent is mapped to an intronic region of Hs.133107. Analysis of expansion, as it has been found in two locations on chro- cross-species chained alignments revealed that sequences mosome 13 as well as on chromosomes 18 and Y (Table homologous to AI792557 can be found in the genomes of 3). the cow, dog, rat, mouse, rhesus, and chimpanzee with similarities of 60% to 72% in non-primate genomes and Cluster Hs. 133294 corresponds to mRNA IQGAP3, of 93% in rhesus and 98% in chimpanzee genomes (Table which encodes a member of the Rho GTPase family of reg- 2). Chimpanzee chromosome 8 contains a 400-bp region ulators involved in cytokinesis. Specifically, cluster Hs. with a near-perfect homology to human EST AI792557, 133294 includes an alternatively spliced isoform of the almost completely overlapping the 344-bp sequence of IQGAP3 gene that arises by retention of its 672-nt intron. interest [see Additional file 2]. We also found an explana- Earlier [4], we demonstrated that this isoform is character- tion for the reported absence of PCR signal in the chim- ized by broad tumor-related and embryonal expression, panzee genome: the AI792557-specific forward primer is thus representing a new carcinoembryonic transcript. disrupted by TTATC deletion located at the border of the segment of human-chimpanzee homology. It is of interest The AL040372-specific sequence corresponding to the that both human and chimpanzee AI792557-like loci are tumor-related transcript of interest is mapped to the 3'-ter- 5'-flanked by an imperfect poly(t) repeat. Genomic minal intron and the 3'-UTR of IQGAP3 mRNA. sequences upstream of the poly(t) repeat and downstream Sequences with strong homology to this genomic region of the 3' end of human-chimpanzee homologous seg- are present in macaque (94%) and chimpanzee (99%) ments do not possess any resemblance. Similarly, genomes. Moreover, sequences with a similarity of 52%– sequences corresponding to AI792557 were found in the 73% to this genomic region have been found in opossum, genome of Macaca mulatta, via BLAT and chained align- mouse, rat, dog, and cow genomes (Table 2). Interest- ments, but not in PCR experiments. Sequences that ingly, the part of the 3'-UTR exonic sequence which is belong to the Hs. 128594 cluster represent human mRNA overexpressed in human tumors according to UniGene CACNA2D3 encoding for the voltage-dependent calcium data is not present (or is highly divergent) in the mouse channel protein alpha 2/delta 3 subunit. At the same time, genome [see Additional file 2]. our target 415-bp sequence, AI952931, is located in an intron of the CACNA2D3 gene. This EST has two exons Summary data of the cross-species homology analysis of and is transcribed from the strand opposite to the gene, as ESTs are presented in Table 2. Similar results were follows from direction of its splice sites consensus. The obtained when experimentally studied PCR fragments genomic sequence corresponding to the 315-bp 3'-exon were analysed [see Additional file 2]. can be found in genomes of cow, dog, rat, and mouse with a similarity of 64%–75%, and with almost perfect identity Using BLAT, we found that AL040372- and AA166653- (93% and 99%) in the macaque and chimpanzee homologous sequences have duplicates in the human and genomes ([see Additional file 2], Table 2). At the same nonhuman primate genomes (Table 3). time, sequences homologous to the 200-bp 3'-terminal fragment of this EST are found in the genomes of opos- Molecular phylogenetic analysis sum (72% similarity) and chicken (61% similarity, Table Fig. 1a represents a phylogeny of AL040372-homologous 2). Only 14% and 16% of the human genomic sequence sequences. The scale bar indicates the relative amount of can be aligned with chicken and opossum orthologs, change along branches. All-against-all BLAT searches respectively (Table 2). Interestingly, the 120-bp sequence among non-primate species sequences were conducted. representing the 5'-exon sequence of AI952931 is entirely Sequences with more than 70% identity were found in absent in all known genomes except human and cow and dog genomes. These sequences were included in macaque. the phylogeny reconstruction. All primates except lemurs produce a well-supported monophyletic group which A 450-bp tumor-related sequence corresponding to the organizes a separate cluster on the phylogenetic tree. non-coding cluster Hs.426704 (former Hs. 154173, core Among these fourteen sequences, lemur, dog, and cow EST AA166653) is mapped to a human ribosomal DNA form separate nodes. Fig. 1b shows a phylogenetic tree of Page 3 of 10 (page number not for citation purposes) Table 2: Summary of cross-species homology analysis results Sequence/Cluster Human Position Compared Aligned Bases % of Aligned Matched Bases % of Matched Human Sequence Compared Sequence Genomes where between Bases** between Bases*** Homology was Genomes Genomes found* Full length of Unmatched Full length of Unmatched Aligned Sequence Bases Aligned Sequence Bases AL040372/Hs.133294 chr1:153 308 opossum 965 88.8 505 52.3 1087 582 2068 1563 314-153 mouse 753 69.3 537 71.3 550 771 234 309 400 rat 1011 93.0 621 61.4 466 895 274 cow 964 88.7 701 72.7 386 1916 1215 dog 1060 97.5 714 67.4 373 1229 515 rhesus 1086 99.9 1018 93.7 69 1073 55 chimpanzee 1086 99.9 1071 98.6 16 1077 6 AI792557/HS.133107 chr8:129 160 mouse 401 80.7 242 60.3 497 255 576 334 366-129 rat 412 82.9 249 60.4 248 561 312 160 862 cow 418 84.1 279 66.7 218 405 126 dog 414 83.3 297 71.7 200 624 327 rhesus 496 99.8 460 92.7 37 488 28 chimpanzee 496 99.8 486 98.0 11 490 4 AA166653/Hs.426704 chr2:132 864 310- rhesus 516 86.0 454 88.0 600 146 712 258 132 864 909 chimpanzee 599 99.8 594 99.2 6603 9 AI952931/HS. 128594 chr3:54 641 chicken 227 14.0 138 60.8 1624 1486 555 417 157-54 opossum 256 15.8 185 72.3 1439 262 77 642 780 mouse 446 27.5 308 69.1 1316 439 131 rat 658 40.5 420 63.8 1204 624 204 cow 1330 81.9 980 73.7 644 1415 435 dog 1302 80.2 977 75.0 647 1318 341 rhesus 1623 99.9 1516 93.4 108 1635 119 chimpanzee 1431 88.1 1413 98.7 211 1432 19 * Fugu, tetraodon, zebrafish, frog, chicken, rat, mouse, cow, dog, macaque, and chimpanzee genomes were analyzed. ** Percent of aligned bases were estimated as the ratio of aligned bases between genomes and the full length of the aligned human sequence. *** Percent of matched bases were estimated as the ratio of matched and aligned bases between genomes. Infectious Agents and Cancer 2006, 1:8 http://www.biomedcentral.com/1750-9378/1/8 Page 4 of 10 (page number not for citation purposes) Infectious Agents and Cancer 2006, 1:8 http://www.biomedcentral.com/1750-9378/1/8 Table 3: Duplications of tumour-related sequences studied in primate genomes Mapping/Transcript (EST Cluster) #1 #2 #3 #4 Original transcript mapping on chromosome in human genome 1 2 3 8 Human duplications and their mapping 1 (13)* 5 (12, 16, Y × 3) 0 0 The number of homologs in P. troglodytes genome and their mapping 2 (1, 14) 4 (13 × 2, 18, Y) 1 (2) 1 (7) The number of homologs in M. mulatta genome** 2 6 1 1 * Chromosomes with sequence duplications are in brackets ** Mapping not shown as M. mulatta genome is available in draft version only. #1-AL040372, Hs.133294; #2-AA166653, Hs.426704; #3-AI952931, Hs.128594; #4-AI792557, Hs.133107 AA166653-homologous sequences. These sequences were ization experiments. However, comparative genomics found in humans but in only four other primates. BLAT analysis has not revealed AA166653-homologous searches against non-primate genomes did not reveal any sequences in the chicken genome. We suggest that this sig- homologies. Pongo sequences form a distinct node. The nal may be an artifact of hybridization. phylogeny of AI792557-homologous sequences among primates is described in Fig. 1c. There are two separate Interestingly, in the case of the AA166653-homologous clusters on the phylogenetic tree, one of which consists of sequence, signals on Southern blot form a "ladder" [see new world monkeys (Ateles and Callimico), and the other Additional file 1], which is a feature of fragments located of which includes apes and old world monkeys. Erythroce- in a repetitive sequence. It is in good agreement with com- bus and macaques, which belong to the old world mon- putational evidence that the AA166653-specific sequence keys, form a separate branch. This phylogenetic tree is located in an intergenic spacer upstream of the 23 repeat corresponds well with the existing classification of pri- region of the human ribosomal DNA complete repeating mates. Fig. 1d represents the phylogeny of AI952931- unit [4], which is tandemly repeated and forms arrays in homologous sequences. Sequences with near 80% simi- genomes of eukaryotes. larity were found in the dog and cow genomes using BLAT. These sequences were included in the phylogeny Comparative genomics analysis have shown that the reconstruction. On this tree, primate sequences formed a tumor-related transcripts under consideration have separate cluster that splits from the dog and cow node. orthologs in mammal genomes only and not in those of Other algorithms (ML and MP) provide similar results in fishes, amphibia, and birds, with the single exception of a trees topology. short sequence in the chicken genome with low homol- ogy for AI952931 (Tables 2 and S1). In some primates (Callimico goeldii, Ateles fusciceps), Alu sequences were found in AL040372-homologous frag- The reason why the probe did not hybridize with DNA ments (Fig. 2a, lanes 2 and 3). These Alu sequences belong from mammals in which we found homologous to type Y, as shown by sequencing (data not shown). Fig. sequences using comparative genomics analysis is due to 2b shows the location of the insert on the genetic map of low homology and the short length of orthologous the IQGAP gene. sequences (Table 2). We may conclude that Southern hybridyzation and com- Discussion The prediction that evolutionarily new sequences may be parative genomics data confirm the evolutionary novelty expressed in tumor cells was made in our previous articles of the sequences studied, i.e., their origins in mammals or [1,2]. To experimentally examine this prediction, we per- in primates. formed Southern hybridization of [α- P]-labeled newly described tumor-related fragments with genomic DNA The results of molecular phylogenetic analysis are in from different animal species. Sequences studied in the accordance with Southern hybridization and comparative present article were selected from tumor-related tran- genomics results. AA166653-homologous sequences are scripts revealed by an in silico search and experimentally present only in apes and macaques and have no homol- described in our previous papers [3,4]. ogy with any sequences in other mammals. The most archaic of the four species presented on the phylogenetic Hybridization signals were detected only with human and tree in Fig. 1b is the macaque. We cannot find an orangutan DNA, with the single exception of a signal AA166653-specific sequence in primates before the diver- observed after hybridization of the AA166653-specific [α- gence of old world monkeys and apes. Therefore, the ori- P]-labeled probe with chicken DNA [see Additional file gin of AA166653-specific sequences took place about 25 1]. This signal was consistently observed in several hybrid- mya, during the divergence of macaques and apes. Page 5 of 10 (page number not for citation purposes) Infectious Agents and Cancer 2006, 1:8 http://www.biomedcentral.com/1750-9378/1/8 Phylog Figure 1 eny trees of tumour-related sequences in primates Phylogeny trees of tumour-related sequences in primates. Trees were constructed with the neighbor-joining method using pairwise deletion and tested with 1,000 bootstrap replicates, (a) Phylogeny of the AL040372-homologous sequence, which demonstrates a divergence of 8% ± 1.4% between Homo sapiens and Lemur catta. (b) Phylogeny analysis of the AA166653-homologous sequence. The maximum divergence in this cluster between Homo sapiens and Pongo pygmaeus is 7.8% ± 1.1%, and the divergence between Homo sapiens and Pan troglodytes is 4% ± 0.3%. (c) Phylogeny of the AI792557-homologous sequence among primates. The maximum divergence between Homo sapiens and Ateles fusciceps is 14.6% ± 1.3% for this sequence, (d) Phylogeny of the AI952931-homologous sequence among primates. The sequence divergence ranges from 0.9% to 7.8%. It was found that Homo sapiens and Pan troglodytes have 1.2% ± 0.4% divergence; the divergence between Homo sapiens and Callimico is 7.8% ± 1.2%. AL040372-, AI792557- and AI952931-specific sequences PVT1, encoding for the Pvt1 oncogene homolog. The Pvt1 formed separate clusters on phylogenetic trees demon- locus also is a common integration site for murine leuke- strating high nucleotide sequence divergence (from 20% mia viruses on mouse chromosome 15 and is located to 35%) with related sequences in mammals (Fig. 1a,1c approximately 270 kb from c-myc. MLV proviruses inte- and 1d). AL040372-homologous sequences were found grated in the Pvt1 locus activate c-myc expression by long- in lemurs – the most archaic members of the primate range (up to-300 kb) cis-effects [6]. In the human genome, group. Lemur sequences demonstrate lower divergence the corresponding sequence is located on chromosome 8. from other primates (about 8%) than related sequences Therefore, an evolutionarily new tumor-specific sequence from non-primate animals (20% and more). Phylogenetic with a high potential of oncogenicity is presented in the analysis has shown that lemur sequences belong to the mammalian lineage near Pvt1 locus. Obvious overexpres- primate phylogenetic cluster. Other primates form a sepa- sion of AI792557-specific transcripts in human tumors rate non-lemur subcluster in this phylogenetic cluster [3,4] could be explained by enhanced transcriptional (Fig. 1a). activity of the c-myc-regulating element. AI792557-homologous sequences form a well-supported The proportion of the mammalian genome which is tran- monophyletic group in apes and old world monkeys. scribed is greater than usually realized [7,8]. It turns out These sequence homologs were found in the Ateles-Callim- that large regions of the genome beyond the coding seg- ico group and were not present in older primates. The ments are transcribed, producing non-coding RNAs divergence of the Ateles-Callimico group from old world [3,7,9]. As shown in this article two of ESTs studied are monkeys took place about 40 mya The cDNA of from introns (plus or minus chains), one from intergenic Hs.133107 which includes EST AI952931, is identified as spacer region and one represent 3-UTR of mRNA, contain- Page 6 of 10 (page number not for citation purposes) Infectious Agents and Cancer 2006, 1:8 http://www.biomedcentral.com/1750-9378/1/8 (a) AL04 Figure 2 0372-specific fragments in a variety of primates (a) AL040372-specific fragments in a variety of primates. The arrow indicates the increase of the fragment size in Ateles and Callimico due to Alu insertion. Lanes: 1, Lemur, 2, Ateles (Alu insertion); 3, Callimico (Alu insertion); 4, Colobus; 5, Eryth- rocebus; 6, Cercopithecus; 7, Macaca; 8, Hylobates; 9, Pongo (Sumatran); 10, Pongo (Bornean); 11, Gorilla (sample 1); 12, Gorilla (sample 2); 13, Pan (sample 1); 14, Pan (sample 2); 15, Homo sapiens. (b) Localization of Alu sequences in the IQGAP gene (Uni- Gene cluster Hs. 133294). ing alternativerly spliced intron. According to our previ- duplications of AL040372- and AA166653-homologous ous data [4], they do not contain easily recognized open sequences in the human, chimpanzee, and macaque reading frames or contain only short open reading frames. genomes (Table 3) supports this interpretation. There is a growing number of recent publications on non- The Alu-Y element was found in a AL040372-homolo- coding RNAs and their possible functions [10-12]. But the gous sequence in Ateles and Callimico. The presence of the fact that certain RNAs have low coding potential may also Alu sequence in the genome may mediate DNA recombi- characterize them as evolving sequences. The concept of nation, the creation of new exons, and the donation of evolution by gene duplication [13] involves understand- new regulatory elements [14]. It was found in our study ing that the extra copy of the duplicated gene may accu- that part of the AL040372-homologous sequence in the mulate mutations and acquire a new function. Before lemur genome has an extension with no similarity in acquisition of a new function, it may express RNA without those of other primates (data not shown). In higher pri- long open reading frames or with stop-codons and/or mates, this region demonstrates a homology with the frame-shift mutations interrupting open reading frames. human genome. In the similar way, non-coding sequences could evolve and eventually acquire a function and/or longer open Taken together, these data from Southern hybridization reading frames. The fact that we were able to demonstrate experiments, molecular phylogenetic studies, and compu- Page 7 of 10 (page number not for citation purposes) Infectious Agents and Cancer 2006, 1:8 http://www.biomedcentral.com/1750-9378/1/8 tational evidence suggest that AA166653-, AL040372-, PCR analysis AI792557- and AI952931-homologous sequences are Oligonucleotide primers for PCR were designed with OLI- indeed evolutionarily new. They originate in mammals GONEW software after alignment of human EST (AA166653 – in primates) and form phylogenetic clusters sequences and the corresponding regions of the human in primates. They are not expressed in normal cells [3,4], genome. We performed BLAST searches for all primer i.e., they are sleeping. pairs created. Only PCR primers that corresponded to a unique location in the human genome and to an EST clus- Earlier [1,2], we formulated the concept of the positive ter of interest were used. evolutionary role of tumors. According to this concept, tumors provide conditions for the expression of evolu- Primers for AA166653: 5'-TCTTTCTTGATGAATTATCT- tionarily new and/or sleeping genes in their cells. As evo- TATG-3' and 5'-ACACACCCTCATTCCCGC-3'; the lutionary new genes we defined genes which participate in expected fragment size is 443 bp. Primers for AL040372: the origin of new cell types [2]. New cell type origin is very 5'-GTCAACCTTCTCATCTTCCTC-3' and 5'-CAGGAAGTT- rare event which is associated with progressive evolution. GGGTAGATGTG-3'; the expected fragment sizes are 412 years of multicellular organisms evolution During 10 bp on cDNA and 1084 bp on genomic DNA. Primers for only about 200 specialized cell types have been originated AI952931: 5'-TAATTGCATTCTTCAAAATTCTAC-3' and 5'- [2]. Thus, within the framework of our hypothesis CTTCGCACCATTGAATAAAC-3'; the expected fragment sequences originated in mammalas may be well consid- size is 315 bp. Primers for AI792557: 5'-TACATAGTTGT- ered as evolutionary new. TATCTTAAGGTG-3' and 5'-TGGGAATTCTATACTTTT- GAC-3'; the expected fragment size is 344 bp. Histone H4 We may guess that during the earliest period of the origin control primers: 5'-ATGTCTGGCCGTGGTAAAGG-3' and of mammals, genome evolution and cellular proliferative 5'-CCGAAGCCGTAAAGAGTGCG-3'; the expected frag- tumor-like processes provided material for the origin of ment size is 300 bp. diversity of mammalian cell and tissue types by generating a diversity of new gene expression patterns. Populations The PCR mixture contained 500 ng of genomic DNA as of tumor-bearing animals could be ancestors of the first template, PCR buffer (1), MgC12 (4 mM), dNTP (each at mammals. Present-day tumors (at the earlier stages of pro- 200 µM), specific forward and reverse primers (each at 0.2 gression) may somehow recapitulate these processes. µM), and Taq DNA Polymerase (1 u) in a total volume of 25 µl (all reagents were supplied by Fermentas, Lithua- Conclusion nia). Our data presented in this and previous articles [3,4] dem- onstrate the expression of relatively evolutionarily new (in PCR was carried out under the following conditions: 1 respect to progressive evolution) and/or sleeping min at 95°C, 35 cycles each consisting of 30 s at 95°C and sequences in tumor cells and support the concept of the 30 s at 56°C for AA166653 primers and histone H4 prim- possible evolutionary role of tumors as a proving ground ers or at 58°C for all other primers, and 1 min at 72°C. At or evolutionary reservoir of expression. If proven to be the final stage of the PCR reaction, mixtures were incu- correct, this concept may substantially increase our capa- bated for 5 min at 72°C to elongate the DNA fragments bilities in the diagnosis and treatment of cancer. This con- synthesized. PCR products were separated by electro- cept may also describe one of the mechanisms of phoresis in 2% agarose gel and visualized by staining with progressive evolution of animal species in which tumors ethidium bromide. participate. Southern hybridization Methods DNA samples were digested with HindIII (10 U per µg of Genomic DNA DNA) for 16 h at 37°C. Digested DNA (8 µg per lane) was Human, ape (Pan troglodytes, Gorilla gorilla, Pongo pyg- electrophoresed in 0.8% agarose gel overnight at 25 V/cm. maeus, Hylobates concolor), old world monkey (Eryth- Gels were stained with ethidium bromide to assess load- rocebus patas, Macaca mulatta, Colobus guereza, ing and blotted onto a nylon membrane, Hybond-N Cercopithecus aethiops), and new world monkey (Cal- (Amersham, USA), according to the manufacturer's limico goeldii, Lemur catta, Ateles fusciceps) genomic instructions. DNAs were used in the study. All samples except human DNA were kindly provided by Dr. S. O'Brien (Chief, Lab- PCR products specific for genes of interest were labeled oratory of Genomic Diversity, National Cancer Institute). with [α- P]dCTP using the HexaLabel DNA Labeling Kit The DNA concentration of each sample was brought to (Fermentas, Lithuania) according to the manufacturer's 200 ng/µl before being used. instructions. Filter prehybridization and hybridization were carried out according to the standard procedure [15]. Page 8 of 10 (page number not for citation purposes) Infectious Agents and Cancer 2006, 1:8 http://www.biomedcentral.com/1750-9378/1/8 Washing conditions were as follows: two times in 0.25 M assessed by bootstrapping with 1,000 replicates (the SEQ- sodium phosphate (pH 7.2), 5% SDS for 30–60 min at BOOT and CONSENCE modules of the PHYLIP). The tree 65°C and two times in 0.125 M sodium phosphate (pH was drawn with Tree View software. 7.2), 1% SDS for 30–60 min at 65°C (medium strin- gency) or two times in 20 mM sodium phosphate (pH Identification of gene duplications and comparative 7.2), 1% SDS for 30–60 min at 65°C (high stringency). X- genomics analysis ray films were exposed to the blots for 3 days at -70°C BLAT searches among primate genome nucleotide with an intensifying screen. sequences were conducted to reveal duplications of sequences under analysis. Matches with a level of identity Cloning and sequencing greater than or equal to 80% of maximum for each Amplified fragments were cloned by standard techniques sequence were taken as duplications. using the bacterial plasmid vector pGEM-T Easy (Promega, USA). Colonies of recombinant DH10B/R E. The cross-species chained alignments database integrated coli cells obtained by electrotransformation were selected. in the Genome Browser tool was used to search for orthol- We subjected recombinant plasmids to restriction endo- ogous sequences in fugu, tetraodon, zebrafish, frog, nuclease analysis and isolated those with fragments of chicken, rat, mouse, cow, dog, macaque, and chimpanzee interest using the Wizard Minipreps Plasmid DNA Purifi- genomes [17]. cation System (Promega, USA). Multiple clone sequenc- ing was performed for each amplicon. Competing interests The author(s) declare that they have no competing inter- Sequencing was carried out by the Sanger method using ests. the AutoCycle Sequencing Kit (Pharmacia Biotech, Swe- den) and standard Cy5-labeled primers T7, whose bind- Authors' contributions ing sites flank the cloning site of recombinant fragment. A.P.K. is an author of original hypothesis of the evolution- We analyzed the products of the sequence reaction with ary role of tumor and general design of experiments. He an automated sequencer, ALFexpress (Pharmacia Biotech, also directed the whole research. D.P and L.L.K. per- Sweden), using the ALFwin v. 1.10 software package formed PCR and Southern experiments. I.D. carried out (Pharmacia Biotech, Sweden). sequencing and molecular phylogenetic analysis. Y.G. and N.S. conducted comparative genomics and bioinformatics Molecular phylogenetic analysis analyses. A.B. participated in the intial stage of bioinfor- PCR amplified fragments of primate DNA were cloned as matics analysis. All authors discussed the results and com- described above. A plasmid collection from each primate mented on the manuscript. was created. In total, 86 clones containing sequences of interest were obtained. For each fragment, at least two Additional material clones were sequenced in forward and reverse directions in order to exclude PCR and sequencing errors. The Additional file 1 BioEdit software was used to generate sequence align- Supplementary Information. The file contains details about Southern ments. The alignments consist of the following numbers hybridization analysis results (Figures S1 and S2). of phylogenetically informative sites: 412 for the Click here for file AL040372 fragment, 443 for the AA166653 fragment, 315 [http://www.biomedcentral.com/content/supplementary/1750- 9378-1-8-S1.doc] for the AI952931 fragment, and 344 for the AI792557 fragment. We constructed phylogenetic trees using the Additional file 2 neighbor-joining method. Distance-based reconstruc- Supplementary Information. Table S1. Cross-species alignment results tions and parsimony reconstructions based on the opti- for sequences within experimentally analyzed PCR fragments. Figures mal alignments gave qualitatively similar phylogenetic S3a, S4a, S5a, and S6a show BioEdit alignments of human genome results, with the same major clades and topological differ- sequences corresponding to fragments of interest with homologous ences in nodes. The results of phylogenetic analysis are sequences in the mouse and chimpanzee genomes. In Fig. S6a, homologs presented in Fig. 1. from cow and dog genomes are added. Primers that had been used for RT- PCR experiments are shown. The graphical alignments of the ESTs under study (set in the light yellow box in the pictures) with the human genome Sequencing data were analyzed with the DNASIS v. 2.5 are present in Figures S3b, S4b, S5b, and S6b. Alignments of human software (Hitachi Software Engineering, USA). We carried genome fragments with corresponding genome fragments of other organ- out alignments using the BioEdit software and excluded isms (fugu, tetraodon, zebrafish, frog, chicken). gap-containing sites. Phylogenetic trees were built accord- Click here for file [http://www.biomedcentral.com/content/supplementary/1750- ing to the neighbor-joining method using the Kimura dis- 9378-1-8-S2.doc] tances by the DNADIST and NEIGHBOR modules of the PHYLIP software package and PHYLIP v.3.57c [16], respectively. The reliability of the tree topology was Page 9 of 10 (page number not for citation purposes) Infectious Agents and Cancer 2006, 1:8 http://www.biomedcentral.com/1750-9378/1/8 Acknowledgements The authors thank S. O'Brien for the primate DNA panel and V. Evtush- enko for discussions. This results were presented: • As lecture on National Cancer Institute/Center for Cancer Research Grand Rounds (November 2, 2004). • As oral abstract «Expression of Evolutionary New Sequences in Human tumors», MBE 05 Conference, Allan Wilson Centre for Molecular Ecology and Evolution, University of Auckland, Auckland, New Zealand, 2005. • As plenary lecture, «In silico gel hybridization for tumor Ag discovery», International Cancer Vaccine Conference, May 26–28, 2006, Naples, Italy References 1. Kozlov AP: Evolution of Living Organisms as a Multilevel Process. J Theor Biol 1979, 81:1. 2. Kozlov AP: Gene Competition and the Possible Evolutionary Role of Tumours. Medical Hypotheses 1996, 46:81. 3. Baranova AV, Lobashev AV, Ivanov DV, Krukovskaya LL, Yankovsky NK, Kozlov A: In silico screening for tumour-specific expressed sequences in human genome. FEBS Lett 2001, 508:143. 4. Krukovskaja LL, Baranova AV, Tyezelova T, Polev D, Kozlov AP: Experimental study of human expressed sequences newly identified in silico as tumour specific. Tumour Biol 2005, 26:17. 5. Napier JR, Napier PH: The Natural History of The Primates MIT Press, Cambridge, MA; 1985. 6. Koehne CF, Lazo PA, Alves K, Lee JS, Tsichlis PN, O'Donnell PV: The Mlvi-1 locus involved in the induction of rat T-cell lympho- mas and the pvt-1/Mis-1 locus are identical. J Virol 1989, 63:2366. 7. Kapranov P, Cawley SE, Drenkow J, Bekiranov S, Strausberg RL, Fodor SP, Gingeras TR: Large-Scale Transcriptional Activity in Chromosomes 21 and 22. Science 2002, 296:916. 8. Evtushenko VI, Hanson KP, Barabitckaya OV, Emelyanov AV, Reshet- nikov VL, Kozlov AP: Determination of the upper level of expression of mammalian genome. Mol Biol (Mosk) 1989, 23:663. 9. Nekrutenko A: Reconciling the numbers: ESTs versus protein- coding genes. Mol Biol Evol 2004, 21:1278. 10. Kelley RL, Kuroda MI: Noncoding RNA Genes in Dosage Com- pensation and Impriting. Cell 2000, 103:9. 11. Erdmann VA, Barciszewska MZ, Hochberg A, de Groot N, Barcisze- wski J: Regulatory RNAs. Cell Mol Cell Mol Life Sci 2001, 58:960. 12. Huttenhofer A, Kiefmann M, Meier-Ewert S, O'Brien J, Lehrach H, Bachellerie JP, Brosius J: RNomics: an experimental approach that identifies 201 candidates for novel, small, non-messen- ger RNAs in mouse. EMBO J 2001, 20:2943. 13. Ohno S: Evolution by Gene Duplication Springer-Verlag, New York; 14. Batzer M, Deininger P: Alu repeats and human genomic diver- sity Alu repeats and human genomic diversity. Nature Rev Genet 2002, 3:370. 15. Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A Laboratory Publish with Bio Med Central and every Manual Cold Spring Harbor, New York; 1989. scientist can read your work free of charge 16. PHYLIP [http://evolution.genetics.washington.edu/phylip.html] 17. Kent WJ, Baertsch R, Hinrichs A, Miller W, Haussler D: Evolution's "BioMed Central will be the most significant development for cauldron: Duplication, deletion, and rearrangement in the disseminating the results of biomedical researc h in our lifetime." mouse and human genomes. Proc Natl Acad Sci USA 2003, Sir Paul Nurse, Cancer Research UK 100:11484. 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Springer Journals
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Copyright © 2006 by Kozlov et al; licensee BioMed Central Ltd.
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Biomedicine; Cancer Research; Infectious Diseases; Oncology
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1750-9378
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10.1186/1750-9378-1-8
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17189608
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Abstract

Background: Earlier we suggested the concept of the positive evolutionary role of tumors. According to this concept, tumors provide conditions for the expression of evolutionarily new and/ or sleeping genes in their cells. Thus, tumors are considered as evolutionary proving ground or reservoir of expression. To support this concept we have previously characterized in silico and experimentally a new class of human tumor-related transcribed sequences. Results: In this article we describe results of further studies of previously described tumor-related sequences. The results of molecular phylogeny studies, Southern hybridization experiments and computational comparison with genomes of other species are presented. Conclusion: These results suggest that these previously described tumor-related human transcripts are also relatively evolutionarily new. Background i.e., the tumor-specificity of expression of these sequences In previous studies [1,2], we formulated the concept of [4]. the positive evolutionary role of tumors. According to this concept, tumors provide conditions for the expression of To experimentally examine our prediction [1,2] that at evolutionarily new and/or sleeping genes in their cells. least some tumor-related sequences are evolutionarily Thus, tumors are considered as an evolutionary proving new, we performed Southern hybridization of our newly ground of expression. described tumor-related sequences with genomic DNA from different animal species. Hybridization was found In earlier work using the computational differential dis- only with human and orangutan DNA, with one excep- play approach, we identified a considerable number of tion in which a signal was also developed with chicken human tumor-related expressed sequence tag (EST) clus- DNA. ters many of which had not been described previously [3]. Experimental data confirmed the results obtained in silico, We performed a search for ortholog sequences in fugu, tetraodon, zebrafish, frog, chicken, rat, mouse, cow, dog, Page 1 of 10 (page number not for citation purposes) Infectious Agents and Cancer 2006, 1:8 http://www.biomedcentral.com/1750-9378/1/8 macaque, and chimpanzee genomes using cross-species Bank:AL040372], now in cluster Hs.133294; chained alignments. This search confirmed that our newly [GenBank:AI952931] from cluster Hs.128594 (former ] from cluster described tumor-related transcripts are relatively evolu- Hs.67624); and [GenBank:AI792557 tionarily new, with some of their orthologs having origi- Hs.133107. nated in mammals and others in primates. PCR analysis and Southern hybridization PCR experiments with specific primers were performed on We performed Southern hybridization of [α- P]-labeled a panel of DNAs from different primates. Amplified frag- sequence-specific fragments with genomic DNA from ments were cloned and sequenced, and their molecular eleven different animal species: lamprey, fish, frog, phylogeny was studied. The results show that these chicken, pigeon, mouse, rat, guinea pig, sheep, horse, and sequences form well-defined phylogenetic clusters which human. Southern hybridization analysis reveals only correspond to the phylogeny of primates as previously homology sequences in chicken genome for AA166653- understood. specific probe. In addition, we were able to demonstrate by Southern hybridization that a sequence homologous Taken together, our Southern hybridization, molecular to the human AA166653-specific 11.2-kb fragment is phylogeny, and comparative genomics data support our present in orangutan DNA [see Additional file 1]. prediction [1,2] that evolutionarily new and/or sleeping sequences may be specifically expressed in tumor cells. Therefore, we performed PCR amplification of sequence- specific fragments on the panel of primate DNAs. The results of PCR experiments and comparative genomics Results Transcribed sequences analyzed data obtained by homology analysis of these tumor- Because of the constant rebuilding of the UniGene clus- related sequences within primate DNA are presented in ters and EST shuffling between them, we cannot follow Table 1. As follows from the results shown in Table 1, the history of each cluster. Clusters very often do not mark sequences homologous to tumor-related human EST are a specific transcript, but a set of transcripts whose genome found in a variety of primates. mapping regions are often neighboring but may be not overlapping. Therefore, we selected ESTs which were used AL040372- and AI792557-specific tumor-related human for primer design in our previous investigations [3,4] and sequences are found in the majority of primate species followed the history of their sequences in UniGene. studied, even in the most archaic. The AI792557-homolo- gous sequence is not found in lemurs and colobus mon- In this paper, we present analyses of the following ESTs keys. The AA166653-homologous sequence is present (UniGene buid 185): [GenBank:AA166653], now in clus- only in apes and macaques. The AI952931-homologous ter Hs.426704 (former Hs. 154173); [Gen- sequence is found in apes, new world monkeys, and Table 1: Results of PCR experiments* and comparative genomics analysis within primates. Superfamily Species/Transcript (EST Clusters) #1 #2 #3 #4 Platyrrhini Lemur catta + --- New World monkeys Ateles fusciceps + - + + Callimico goeldii + - + + Cercopitecoidea Colobus guereza + --- Old World monkeys Erythrocebus patas + - - + Cercopithecus aethiops + - - + Macaca mulatta (+)** (+) (+) (+) Hominoidea Hylobates concolor + - + + Apes and Human Pongo pygmaeus (sumatran) + + + + Pongo pygmaeus (bornean) -- + + Gorilla gorilla (sample 1) + + + + Gorilla gorilla (sample 2) -- +/-*** + Pan troglodytes (sample 1) + + + (+) Pan troglodytes (sample 2) + + + (+) Homo sapiens + + + + * DNA samples were ranged according to the existing classification of primates [5]. ** Data in brackets are results of comparative genomics analysis. *** Weak signal. #1-AL040372, Hs.133294; #2-AA166653, Hs.426704; #3-AI952931, Hs.128594; #4-AI792557, Hs.133107 Page 2 of 10 (page number not for citation purposes) Infectious Agents and Cancer 2006, 1:8 http://www.biomedcentral.com/1750-9378/1/8 macaques. No sequences discussed in this article could be complete repeating unit. For this cluster, we used PCR amplified by PCR on a DNA evolutionary panel with primers specific to sequences located on chromosome 2. genomic DNA from species non-primate species using the According to our experimental data [4], this sequence is selected primers. expressed in carcinomas only. This sequence has not been found in any sequenced mammalian genome except rhe- Comparative genomics and bioinformatics analyses sus and chimpanzee, with similarity levels of 88% and Hs.133107 cluster consists of spliced mRNAs, but the 99%, respectively ([see Additional file 2], Table 2). In the studied sequence AI792557 is a short, unspliced EST and chimpanzee genome, the Hs.426704 locus underwent is mapped to an intronic region of Hs.133107. Analysis of expansion, as it has been found in two locations on chro- cross-species chained alignments revealed that sequences mosome 13 as well as on chromosomes 18 and Y (Table homologous to AI792557 can be found in the genomes of 3). the cow, dog, rat, mouse, rhesus, and chimpanzee with similarities of 60% to 72% in non-primate genomes and Cluster Hs. 133294 corresponds to mRNA IQGAP3, of 93% in rhesus and 98% in chimpanzee genomes (Table which encodes a member of the Rho GTPase family of reg- 2). Chimpanzee chromosome 8 contains a 400-bp region ulators involved in cytokinesis. Specifically, cluster Hs. with a near-perfect homology to human EST AI792557, 133294 includes an alternatively spliced isoform of the almost completely overlapping the 344-bp sequence of IQGAP3 gene that arises by retention of its 672-nt intron. interest [see Additional file 2]. We also found an explana- Earlier [4], we demonstrated that this isoform is character- tion for the reported absence of PCR signal in the chim- ized by broad tumor-related and embryonal expression, panzee genome: the AI792557-specific forward primer is thus representing a new carcinoembryonic transcript. disrupted by TTATC deletion located at the border of the segment of human-chimpanzee homology. It is of interest The AL040372-specific sequence corresponding to the that both human and chimpanzee AI792557-like loci are tumor-related transcript of interest is mapped to the 3'-ter- 5'-flanked by an imperfect poly(t) repeat. Genomic minal intron and the 3'-UTR of IQGAP3 mRNA. sequences upstream of the poly(t) repeat and downstream Sequences with strong homology to this genomic region of the 3' end of human-chimpanzee homologous seg- are present in macaque (94%) and chimpanzee (99%) ments do not possess any resemblance. Similarly, genomes. Moreover, sequences with a similarity of 52%– sequences corresponding to AI792557 were found in the 73% to this genomic region have been found in opossum, genome of Macaca mulatta, via BLAT and chained align- mouse, rat, dog, and cow genomes (Table 2). Interest- ments, but not in PCR experiments. Sequences that ingly, the part of the 3'-UTR exonic sequence which is belong to the Hs. 128594 cluster represent human mRNA overexpressed in human tumors according to UniGene CACNA2D3 encoding for the voltage-dependent calcium data is not present (or is highly divergent) in the mouse channel protein alpha 2/delta 3 subunit. At the same time, genome [see Additional file 2]. our target 415-bp sequence, AI952931, is located in an intron of the CACNA2D3 gene. This EST has two exons Summary data of the cross-species homology analysis of and is transcribed from the strand opposite to the gene, as ESTs are presented in Table 2. Similar results were follows from direction of its splice sites consensus. The obtained when experimentally studied PCR fragments genomic sequence corresponding to the 315-bp 3'-exon were analysed [see Additional file 2]. can be found in genomes of cow, dog, rat, and mouse with a similarity of 64%–75%, and with almost perfect identity Using BLAT, we found that AL040372- and AA166653- (93% and 99%) in the macaque and chimpanzee homologous sequences have duplicates in the human and genomes ([see Additional file 2], Table 2). At the same nonhuman primate genomes (Table 3). time, sequences homologous to the 200-bp 3'-terminal fragment of this EST are found in the genomes of opos- Molecular phylogenetic analysis sum (72% similarity) and chicken (61% similarity, Table Fig. 1a represents a phylogeny of AL040372-homologous 2). Only 14% and 16% of the human genomic sequence sequences. The scale bar indicates the relative amount of can be aligned with chicken and opossum orthologs, change along branches. All-against-all BLAT searches respectively (Table 2). Interestingly, the 120-bp sequence among non-primate species sequences were conducted. representing the 5'-exon sequence of AI952931 is entirely Sequences with more than 70% identity were found in absent in all known genomes except human and cow and dog genomes. These sequences were included in macaque. the phylogeny reconstruction. All primates except lemurs produce a well-supported monophyletic group which A 450-bp tumor-related sequence corresponding to the organizes a separate cluster on the phylogenetic tree. non-coding cluster Hs.426704 (former Hs. 154173, core Among these fourteen sequences, lemur, dog, and cow EST AA166653) is mapped to a human ribosomal DNA form separate nodes. Fig. 1b shows a phylogenetic tree of Page 3 of 10 (page number not for citation purposes) Table 2: Summary of cross-species homology analysis results Sequence/Cluster Human Position Compared Aligned Bases % of Aligned Matched Bases % of Matched Human Sequence Compared Sequence Genomes where between Bases** between Bases*** Homology was Genomes Genomes found* Full length of Unmatched Full length of Unmatched Aligned Sequence Bases Aligned Sequence Bases AL040372/Hs.133294 chr1:153 308 opossum 965 88.8 505 52.3 1087 582 2068 1563 314-153 mouse 753 69.3 537 71.3 550 771 234 309 400 rat 1011 93.0 621 61.4 466 895 274 cow 964 88.7 701 72.7 386 1916 1215 dog 1060 97.5 714 67.4 373 1229 515 rhesus 1086 99.9 1018 93.7 69 1073 55 chimpanzee 1086 99.9 1071 98.6 16 1077 6 AI792557/HS.133107 chr8:129 160 mouse 401 80.7 242 60.3 497 255 576 334 366-129 rat 412 82.9 249 60.4 248 561 312 160 862 cow 418 84.1 279 66.7 218 405 126 dog 414 83.3 297 71.7 200 624 327 rhesus 496 99.8 460 92.7 37 488 28 chimpanzee 496 99.8 486 98.0 11 490 4 AA166653/Hs.426704 chr2:132 864 310- rhesus 516 86.0 454 88.0 600 146 712 258 132 864 909 chimpanzee 599 99.8 594 99.2 6603 9 AI952931/HS. 128594 chr3:54 641 chicken 227 14.0 138 60.8 1624 1486 555 417 157-54 opossum 256 15.8 185 72.3 1439 262 77 642 780 mouse 446 27.5 308 69.1 1316 439 131 rat 658 40.5 420 63.8 1204 624 204 cow 1330 81.9 980 73.7 644 1415 435 dog 1302 80.2 977 75.0 647 1318 341 rhesus 1623 99.9 1516 93.4 108 1635 119 chimpanzee 1431 88.1 1413 98.7 211 1432 19 * Fugu, tetraodon, zebrafish, frog, chicken, rat, mouse, cow, dog, macaque, and chimpanzee genomes were analyzed. ** Percent of aligned bases were estimated as the ratio of aligned bases between genomes and the full length of the aligned human sequence. *** Percent of matched bases were estimated as the ratio of matched and aligned bases between genomes. Infectious Agents and Cancer 2006, 1:8 http://www.biomedcentral.com/1750-9378/1/8 Page 4 of 10 (page number not for citation purposes) Infectious Agents and Cancer 2006, 1:8 http://www.biomedcentral.com/1750-9378/1/8 Table 3: Duplications of tumour-related sequences studied in primate genomes Mapping/Transcript (EST Cluster) #1 #2 #3 #4 Original transcript mapping on chromosome in human genome 1 2 3 8 Human duplications and their mapping 1 (13)* 5 (12, 16, Y × 3) 0 0 The number of homologs in P. troglodytes genome and their mapping 2 (1, 14) 4 (13 × 2, 18, Y) 1 (2) 1 (7) The number of homologs in M. mulatta genome** 2 6 1 1 * Chromosomes with sequence duplications are in brackets ** Mapping not shown as M. mulatta genome is available in draft version only. #1-AL040372, Hs.133294; #2-AA166653, Hs.426704; #3-AI952931, Hs.128594; #4-AI792557, Hs.133107 AA166653-homologous sequences. These sequences were ization experiments. However, comparative genomics found in humans but in only four other primates. BLAT analysis has not revealed AA166653-homologous searches against non-primate genomes did not reveal any sequences in the chicken genome. We suggest that this sig- homologies. Pongo sequences form a distinct node. The nal may be an artifact of hybridization. phylogeny of AI792557-homologous sequences among primates is described in Fig. 1c. There are two separate Interestingly, in the case of the AA166653-homologous clusters on the phylogenetic tree, one of which consists of sequence, signals on Southern blot form a "ladder" [see new world monkeys (Ateles and Callimico), and the other Additional file 1], which is a feature of fragments located of which includes apes and old world monkeys. Erythroce- in a repetitive sequence. It is in good agreement with com- bus and macaques, which belong to the old world mon- putational evidence that the AA166653-specific sequence keys, form a separate branch. This phylogenetic tree is located in an intergenic spacer upstream of the 23 repeat corresponds well with the existing classification of pri- region of the human ribosomal DNA complete repeating mates. Fig. 1d represents the phylogeny of AI952931- unit [4], which is tandemly repeated and forms arrays in homologous sequences. Sequences with near 80% simi- genomes of eukaryotes. larity were found in the dog and cow genomes using BLAT. These sequences were included in the phylogeny Comparative genomics analysis have shown that the reconstruction. On this tree, primate sequences formed a tumor-related transcripts under consideration have separate cluster that splits from the dog and cow node. orthologs in mammal genomes only and not in those of Other algorithms (ML and MP) provide similar results in fishes, amphibia, and birds, with the single exception of a trees topology. short sequence in the chicken genome with low homol- ogy for AI952931 (Tables 2 and S1). In some primates (Callimico goeldii, Ateles fusciceps), Alu sequences were found in AL040372-homologous frag- The reason why the probe did not hybridize with DNA ments (Fig. 2a, lanes 2 and 3). These Alu sequences belong from mammals in which we found homologous to type Y, as shown by sequencing (data not shown). Fig. sequences using comparative genomics analysis is due to 2b shows the location of the insert on the genetic map of low homology and the short length of orthologous the IQGAP gene. sequences (Table 2). We may conclude that Southern hybridyzation and com- Discussion The prediction that evolutionarily new sequences may be parative genomics data confirm the evolutionary novelty expressed in tumor cells was made in our previous articles of the sequences studied, i.e., their origins in mammals or [1,2]. To experimentally examine this prediction, we per- in primates. formed Southern hybridization of [α- P]-labeled newly described tumor-related fragments with genomic DNA The results of molecular phylogenetic analysis are in from different animal species. Sequences studied in the accordance with Southern hybridization and comparative present article were selected from tumor-related tran- genomics results. AA166653-homologous sequences are scripts revealed by an in silico search and experimentally present only in apes and macaques and have no homol- described in our previous papers [3,4]. ogy with any sequences in other mammals. The most archaic of the four species presented on the phylogenetic Hybridization signals were detected only with human and tree in Fig. 1b is the macaque. We cannot find an orangutan DNA, with the single exception of a signal AA166653-specific sequence in primates before the diver- observed after hybridization of the AA166653-specific [α- gence of old world monkeys and apes. Therefore, the ori- P]-labeled probe with chicken DNA [see Additional file gin of AA166653-specific sequences took place about 25 1]. This signal was consistently observed in several hybrid- mya, during the divergence of macaques and apes. Page 5 of 10 (page number not for citation purposes) Infectious Agents and Cancer 2006, 1:8 http://www.biomedcentral.com/1750-9378/1/8 Phylog Figure 1 eny trees of tumour-related sequences in primates Phylogeny trees of tumour-related sequences in primates. Trees were constructed with the neighbor-joining method using pairwise deletion and tested with 1,000 bootstrap replicates, (a) Phylogeny of the AL040372-homologous sequence, which demonstrates a divergence of 8% ± 1.4% between Homo sapiens and Lemur catta. (b) Phylogeny analysis of the AA166653-homologous sequence. The maximum divergence in this cluster between Homo sapiens and Pongo pygmaeus is 7.8% ± 1.1%, and the divergence between Homo sapiens and Pan troglodytes is 4% ± 0.3%. (c) Phylogeny of the AI792557-homologous sequence among primates. The maximum divergence between Homo sapiens and Ateles fusciceps is 14.6% ± 1.3% for this sequence, (d) Phylogeny of the AI952931-homologous sequence among primates. The sequence divergence ranges from 0.9% to 7.8%. It was found that Homo sapiens and Pan troglodytes have 1.2% ± 0.4% divergence; the divergence between Homo sapiens and Callimico is 7.8% ± 1.2%. AL040372-, AI792557- and AI952931-specific sequences PVT1, encoding for the Pvt1 oncogene homolog. The Pvt1 formed separate clusters on phylogenetic trees demon- locus also is a common integration site for murine leuke- strating high nucleotide sequence divergence (from 20% mia viruses on mouse chromosome 15 and is located to 35%) with related sequences in mammals (Fig. 1a,1c approximately 270 kb from c-myc. MLV proviruses inte- and 1d). AL040372-homologous sequences were found grated in the Pvt1 locus activate c-myc expression by long- in lemurs – the most archaic members of the primate range (up to-300 kb) cis-effects [6]. In the human genome, group. Lemur sequences demonstrate lower divergence the corresponding sequence is located on chromosome 8. from other primates (about 8%) than related sequences Therefore, an evolutionarily new tumor-specific sequence from non-primate animals (20% and more). Phylogenetic with a high potential of oncogenicity is presented in the analysis has shown that lemur sequences belong to the mammalian lineage near Pvt1 locus. Obvious overexpres- primate phylogenetic cluster. Other primates form a sepa- sion of AI792557-specific transcripts in human tumors rate non-lemur subcluster in this phylogenetic cluster [3,4] could be explained by enhanced transcriptional (Fig. 1a). activity of the c-myc-regulating element. AI792557-homologous sequences form a well-supported The proportion of the mammalian genome which is tran- monophyletic group in apes and old world monkeys. scribed is greater than usually realized [7,8]. It turns out These sequence homologs were found in the Ateles-Callim- that large regions of the genome beyond the coding seg- ico group and were not present in older primates. The ments are transcribed, producing non-coding RNAs divergence of the Ateles-Callimico group from old world [3,7,9]. As shown in this article two of ESTs studied are monkeys took place about 40 mya The cDNA of from introns (plus or minus chains), one from intergenic Hs.133107 which includes EST AI952931, is identified as spacer region and one represent 3-UTR of mRNA, contain- Page 6 of 10 (page number not for citation purposes) Infectious Agents and Cancer 2006, 1:8 http://www.biomedcentral.com/1750-9378/1/8 (a) AL04 Figure 2 0372-specific fragments in a variety of primates (a) AL040372-specific fragments in a variety of primates. The arrow indicates the increase of the fragment size in Ateles and Callimico due to Alu insertion. Lanes: 1, Lemur, 2, Ateles (Alu insertion); 3, Callimico (Alu insertion); 4, Colobus; 5, Eryth- rocebus; 6, Cercopithecus; 7, Macaca; 8, Hylobates; 9, Pongo (Sumatran); 10, Pongo (Bornean); 11, Gorilla (sample 1); 12, Gorilla (sample 2); 13, Pan (sample 1); 14, Pan (sample 2); 15, Homo sapiens. (b) Localization of Alu sequences in the IQGAP gene (Uni- Gene cluster Hs. 133294). ing alternativerly spliced intron. According to our previ- duplications of AL040372- and AA166653-homologous ous data [4], they do not contain easily recognized open sequences in the human, chimpanzee, and macaque reading frames or contain only short open reading frames. genomes (Table 3) supports this interpretation. There is a growing number of recent publications on non- The Alu-Y element was found in a AL040372-homolo- coding RNAs and their possible functions [10-12]. But the gous sequence in Ateles and Callimico. The presence of the fact that certain RNAs have low coding potential may also Alu sequence in the genome may mediate DNA recombi- characterize them as evolving sequences. The concept of nation, the creation of new exons, and the donation of evolution by gene duplication [13] involves understand- new regulatory elements [14]. It was found in our study ing that the extra copy of the duplicated gene may accu- that part of the AL040372-homologous sequence in the mulate mutations and acquire a new function. Before lemur genome has an extension with no similarity in acquisition of a new function, it may express RNA without those of other primates (data not shown). In higher pri- long open reading frames or with stop-codons and/or mates, this region demonstrates a homology with the frame-shift mutations interrupting open reading frames. human genome. In the similar way, non-coding sequences could evolve and eventually acquire a function and/or longer open Taken together, these data from Southern hybridization reading frames. The fact that we were able to demonstrate experiments, molecular phylogenetic studies, and compu- Page 7 of 10 (page number not for citation purposes) Infectious Agents and Cancer 2006, 1:8 http://www.biomedcentral.com/1750-9378/1/8 tational evidence suggest that AA166653-, AL040372-, PCR analysis AI792557- and AI952931-homologous sequences are Oligonucleotide primers for PCR were designed with OLI- indeed evolutionarily new. They originate in mammals GONEW software after alignment of human EST (AA166653 – in primates) and form phylogenetic clusters sequences and the corresponding regions of the human in primates. They are not expressed in normal cells [3,4], genome. We performed BLAST searches for all primer i.e., they are sleeping. pairs created. Only PCR primers that corresponded to a unique location in the human genome and to an EST clus- Earlier [1,2], we formulated the concept of the positive ter of interest were used. evolutionary role of tumors. According to this concept, tumors provide conditions for the expression of evolu- Primers for AA166653: 5'-TCTTTCTTGATGAATTATCT- tionarily new and/or sleeping genes in their cells. As evo- TATG-3' and 5'-ACACACCCTCATTCCCGC-3'; the lutionary new genes we defined genes which participate in expected fragment size is 443 bp. Primers for AL040372: the origin of new cell types [2]. New cell type origin is very 5'-GTCAACCTTCTCATCTTCCTC-3' and 5'-CAGGAAGTT- rare event which is associated with progressive evolution. GGGTAGATGTG-3'; the expected fragment sizes are 412 years of multicellular organisms evolution During 10 bp on cDNA and 1084 bp on genomic DNA. Primers for only about 200 specialized cell types have been originated AI952931: 5'-TAATTGCATTCTTCAAAATTCTAC-3' and 5'- [2]. Thus, within the framework of our hypothesis CTTCGCACCATTGAATAAAC-3'; the expected fragment sequences originated in mammalas may be well consid- size is 315 bp. Primers for AI792557: 5'-TACATAGTTGT- ered as evolutionary new. TATCTTAAGGTG-3' and 5'-TGGGAATTCTATACTTTT- GAC-3'; the expected fragment size is 344 bp. Histone H4 We may guess that during the earliest period of the origin control primers: 5'-ATGTCTGGCCGTGGTAAAGG-3' and of mammals, genome evolution and cellular proliferative 5'-CCGAAGCCGTAAAGAGTGCG-3'; the expected frag- tumor-like processes provided material for the origin of ment size is 300 bp. diversity of mammalian cell and tissue types by generating a diversity of new gene expression patterns. Populations The PCR mixture contained 500 ng of genomic DNA as of tumor-bearing animals could be ancestors of the first template, PCR buffer (1), MgC12 (4 mM), dNTP (each at mammals. Present-day tumors (at the earlier stages of pro- 200 µM), specific forward and reverse primers (each at 0.2 gression) may somehow recapitulate these processes. µM), and Taq DNA Polymerase (1 u) in a total volume of 25 µl (all reagents were supplied by Fermentas, Lithua- Conclusion nia). Our data presented in this and previous articles [3,4] dem- onstrate the expression of relatively evolutionarily new (in PCR was carried out under the following conditions: 1 respect to progressive evolution) and/or sleeping min at 95°C, 35 cycles each consisting of 30 s at 95°C and sequences in tumor cells and support the concept of the 30 s at 56°C for AA166653 primers and histone H4 prim- possible evolutionary role of tumors as a proving ground ers or at 58°C for all other primers, and 1 min at 72°C. At or evolutionary reservoir of expression. If proven to be the final stage of the PCR reaction, mixtures were incu- correct, this concept may substantially increase our capa- bated for 5 min at 72°C to elongate the DNA fragments bilities in the diagnosis and treatment of cancer. This con- synthesized. PCR products were separated by electro- cept may also describe one of the mechanisms of phoresis in 2% agarose gel and visualized by staining with progressive evolution of animal species in which tumors ethidium bromide. participate. Southern hybridization Methods DNA samples were digested with HindIII (10 U per µg of Genomic DNA DNA) for 16 h at 37°C. Digested DNA (8 µg per lane) was Human, ape (Pan troglodytes, Gorilla gorilla, Pongo pyg- electrophoresed in 0.8% agarose gel overnight at 25 V/cm. maeus, Hylobates concolor), old world monkey (Eryth- Gels were stained with ethidium bromide to assess load- rocebus patas, Macaca mulatta, Colobus guereza, ing and blotted onto a nylon membrane, Hybond-N Cercopithecus aethiops), and new world monkey (Cal- (Amersham, USA), according to the manufacturer's limico goeldii, Lemur catta, Ateles fusciceps) genomic instructions. DNAs were used in the study. All samples except human DNA were kindly provided by Dr. S. O'Brien (Chief, Lab- PCR products specific for genes of interest were labeled oratory of Genomic Diversity, National Cancer Institute). with [α- P]dCTP using the HexaLabel DNA Labeling Kit The DNA concentration of each sample was brought to (Fermentas, Lithuania) according to the manufacturer's 200 ng/µl before being used. instructions. Filter prehybridization and hybridization were carried out according to the standard procedure [15]. Page 8 of 10 (page number not for citation purposes) Infectious Agents and Cancer 2006, 1:8 http://www.biomedcentral.com/1750-9378/1/8 Washing conditions were as follows: two times in 0.25 M assessed by bootstrapping with 1,000 replicates (the SEQ- sodium phosphate (pH 7.2), 5% SDS for 30–60 min at BOOT and CONSENCE modules of the PHYLIP). The tree 65°C and two times in 0.125 M sodium phosphate (pH was drawn with Tree View software. 7.2), 1% SDS for 30–60 min at 65°C (medium strin- gency) or two times in 20 mM sodium phosphate (pH Identification of gene duplications and comparative 7.2), 1% SDS for 30–60 min at 65°C (high stringency). X- genomics analysis ray films were exposed to the blots for 3 days at -70°C BLAT searches among primate genome nucleotide with an intensifying screen. sequences were conducted to reveal duplications of sequences under analysis. Matches with a level of identity Cloning and sequencing greater than or equal to 80% of maximum for each Amplified fragments were cloned by standard techniques sequence were taken as duplications. using the bacterial plasmid vector pGEM-T Easy (Promega, USA). Colonies of recombinant DH10B/R E. The cross-species chained alignments database integrated coli cells obtained by electrotransformation were selected. in the Genome Browser tool was used to search for orthol- We subjected recombinant plasmids to restriction endo- ogous sequences in fugu, tetraodon, zebrafish, frog, nuclease analysis and isolated those with fragments of chicken, rat, mouse, cow, dog, macaque, and chimpanzee interest using the Wizard Minipreps Plasmid DNA Purifi- genomes [17]. cation System (Promega, USA). Multiple clone sequenc- ing was performed for each amplicon. Competing interests The author(s) declare that they have no competing inter- Sequencing was carried out by the Sanger method using ests. the AutoCycle Sequencing Kit (Pharmacia Biotech, Swe- den) and standard Cy5-labeled primers T7, whose bind- Authors' contributions ing sites flank the cloning site of recombinant fragment. A.P.K. is an author of original hypothesis of the evolution- We analyzed the products of the sequence reaction with ary role of tumor and general design of experiments. He an automated sequencer, ALFexpress (Pharmacia Biotech, also directed the whole research. D.P and L.L.K. per- Sweden), using the ALFwin v. 1.10 software package formed PCR and Southern experiments. I.D. carried out (Pharmacia Biotech, Sweden). sequencing and molecular phylogenetic analysis. Y.G. and N.S. conducted comparative genomics and bioinformatics Molecular phylogenetic analysis analyses. A.B. participated in the intial stage of bioinfor- PCR amplified fragments of primate DNA were cloned as matics analysis. All authors discussed the results and com- described above. A plasmid collection from each primate mented on the manuscript. was created. In total, 86 clones containing sequences of interest were obtained. For each fragment, at least two Additional material clones were sequenced in forward and reverse directions in order to exclude PCR and sequencing errors. The Additional file 1 BioEdit software was used to generate sequence align- Supplementary Information. The file contains details about Southern ments. The alignments consist of the following numbers hybridization analysis results (Figures S1 and S2). of phylogenetically informative sites: 412 for the Click here for file AL040372 fragment, 443 for the AA166653 fragment, 315 [http://www.biomedcentral.com/content/supplementary/1750- 9378-1-8-S1.doc] for the AI952931 fragment, and 344 for the AI792557 fragment. We constructed phylogenetic trees using the Additional file 2 neighbor-joining method. Distance-based reconstruc- Supplementary Information. Table S1. Cross-species alignment results tions and parsimony reconstructions based on the opti- for sequences within experimentally analyzed PCR fragments. Figures mal alignments gave qualitatively similar phylogenetic S3a, S4a, S5a, and S6a show BioEdit alignments of human genome results, with the same major clades and topological differ- sequences corresponding to fragments of interest with homologous ences in nodes. The results of phylogenetic analysis are sequences in the mouse and chimpanzee genomes. In Fig. S6a, homologs presented in Fig. 1. from cow and dog genomes are added. Primers that had been used for RT- PCR experiments are shown. The graphical alignments of the ESTs under study (set in the light yellow box in the pictures) with the human genome Sequencing data were analyzed with the DNASIS v. 2.5 are present in Figures S3b, S4b, S5b, and S6b. Alignments of human software (Hitachi Software Engineering, USA). We carried genome fragments with corresponding genome fragments of other organ- out alignments using the BioEdit software and excluded isms (fugu, tetraodon, zebrafish, frog, chicken). gap-containing sites. Phylogenetic trees were built accord- Click here for file [http://www.biomedcentral.com/content/supplementary/1750- ing to the neighbor-joining method using the Kimura dis- 9378-1-8-S2.doc] tances by the DNADIST and NEIGHBOR modules of the PHYLIP software package and PHYLIP v.3.57c [16], respectively. The reliability of the tree topology was Page 9 of 10 (page number not for citation purposes) Infectious Agents and Cancer 2006, 1:8 http://www.biomedcentral.com/1750-9378/1/8 Acknowledgements The authors thank S. O'Brien for the primate DNA panel and V. Evtush- enko for discussions. This results were presented: • As lecture on National Cancer Institute/Center for Cancer Research Grand Rounds (November 2, 2004). • As oral abstract «Expression of Evolutionary New Sequences in Human tumors», MBE 05 Conference, Allan Wilson Centre for Molecular Ecology and Evolution, University of Auckland, Auckland, New Zealand, 2005. • As plenary lecture, «In silico gel hybridization for tumor Ag discovery», International Cancer Vaccine Conference, May 26–28, 2006, Naples, Italy References 1. Kozlov AP: Evolution of Living Organisms as a Multilevel Process. J Theor Biol 1979, 81:1. 2. Kozlov AP: Gene Competition and the Possible Evolutionary Role of Tumours. Medical Hypotheses 1996, 46:81. 3. 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Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A Laboratory Publish with Bio Med Central and every Manual Cold Spring Harbor, New York; 1989. scientist can read your work free of charge 16. PHYLIP [http://evolution.genetics.washington.edu/phylip.html] 17. Kent WJ, Baertsch R, Hinrichs A, Miller W, Haussler D: Evolution's "BioMed Central will be the most significant development for cauldron: Duplication, deletion, and rearrangement in the disseminating the results of biomedical researc h in our lifetime." mouse and human genomes. Proc Natl Acad Sci USA 2003, Sir Paul Nurse, Cancer Research UK 100:11484. Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright BioMedcentral Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp Page 10 of 10 (page number not for citation purposes)

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