Identification of novel breast tumor-specific mutation(s) in the q11.2 region of chromosome 17 by RAPD/AP-PCR fingerprinting

Analysis of genetic instability in breast cancer tissues compared to uninvolved breast tissues from the same individuals by RAPD (random amplified polymorphic DNA)/AP-PCR (arbitrarily primed PCR) fingerprinting using 30 arbitrary primers revealed 190 amplified DNA fragments. Presumably, each of these represents a gene locus in a different region of the genome of breast cancer tissues. Among these amplified DNA fragments, 65 (34.2%) exhibited presence and absence or reductions and enhancements in the intensity in breast cancer tissues compared to uninvolved breast tissues from the same individuals, and 11 amplified DNA fragments (5.7%) represented polymorphisms in the uninvolved human breast tissues. Reductions and enhancements in the intensity of some of the amplified fragments were observed indicating allelic gains or losses in the breast tumor genome compared to the matched uninvolved tissue genome. The presence or absence of some of the amplified DNA fragments were observed in this study indicating homozygous deletions or insertions in the breast tumor DNA compared to the matched uninvolved tissue DNA. Notably, an insertion of a 1270 bp amplified fragment was observed in 81% (17 of 21) of the tumor samples using the primer, OPC04. This amplified fragment resolved into two, 1200 and 1300 bp, single-stranded amplified fragments on the denaturing sequencing gel. This separation into single-stranded fragments suggests that the amplified fragment contains a conformation that is semistable. The 1270 bp amplified fragment localizes to the q11.2 region of chromosome 17. Sequence analysis of this fragment showed a significant DNA base sequence similarity (93%) with one of the breast tumor-specific human EST. The similarity with EST sequences and RT-PCR analysis showed that a part of this amplified fragment is from the coding region of the genome. Any one of the events observed in this study could play an important role in the development of breast cancer or could occur during the clonal expansion of the genetically unstable breast cells.     

Assignment of the human alpha 2-plasmin inhibitor gene (PLI) to chromosome 17, region pter-p12, by PCR analysis of somatic cell hybrids.

The human gene for the alpha 2-plasmin inhibitor (PLI) had been assigned by others to the pericentromeric region of chromosome 18 by in situ hybridization. However, when we used a probe for this gene in our efforts to construct a complete physical map of chromosome 18, we discovered that PLI could be excluded from this chromosome. On the basis of the published PLI sequence, we designed primers to sequences in intron 6 and 7 that direct amplification of a 353-bp fragment that includes the entire exon 7. By using PCR analysis of rodent x human hybrid panels, we have unequivocally assigned the PLI locus to human chromosome 17. With a regional mapping panel, the assignment could be narrowed to region 17pter-p12.     

Molecular evolution of aromatic polyketides and comparative sequence analysis of polyketide ketosynthase and 16S ribosomal DNA genes from various streptomyces species

A 613-bp fragment of an essential ketosynthase gene from the biosynthetic pathway of aromatic polyketide antibiotics was sequenced from 99 actinomycetes isolated from soil. Phylogenetic analysis showed that the isolates clustered into clades that correspond to the various classes of aromatic polyketides. Additionally, sequencing of a 120-bp fragment from the gamma-variable region of 16S ribosomal DNA (rDNA) and subsequent comparative sequence analysis revealed incongruity between the ketosynthase and 16S rDNA phylogenetic trees, which strongly suggests that there has been horizontal transfer of aromatic polyketide biosynthesis genes. The results show that the ketosynthase tree could be used for DNA fingerprinting of secondary metabolites and for screening interesting aromatic polyketide biosynthesis genes. Furthermore, the movement of the ketosynthase genes suggests that traditional marker molecules like 16S rDNA give misleading information about the biosynthesis potential of aromatic polyketides, and thus only molecules that are directly involved in the biosynthesis of secondary metabolites can be used to gain information about the biodiversity of antibiotic production in different actinomycetes.     

Analysis of genomic damage in the mutagen-sensitive mus-201 mutant of Drosophila melanogaster by arbitrarily primed PCR (AP-PCR) fingerprinting.

DNA repair mechanisms are important to maintain the stability of the genome. In Drosophila melanogaster, the mus-201 gene is required in the excision repair process. To study the contribution of the mus-201 gene in the stability of the Drosophila genome, we have used the arbitrarily primed PCR fingerprinting method (AP-PCR). We have analysed the changes in the genomic DNA fingerprints from the progeny of wild-type males crossed with mus-201 repair-deficient or repair-proficient females. After induction of DNA damage with 2-acetylaminofluorene (2-AAF) in the wild-type parental males, quantitative and qualitative differences in the AP-PCR fingerprints were detected between the two crosses, and the estimate of the genomic damage detected by AP-PCR has clearly shown that the mus-201 repair deficiency is associated with an increase of genomic damage. The predominant type of alterations detected by AP-PCR under the mus-201 repair-deficient conditions agree with the results obtained in microsatellite PCR analysis, suggesting that the role of the mus-201 gene, necessary in excision repair, is not associated to the mismatch repair process. The work reported here demonstrates that the AP-PCR is a suitable technique to analyse genetic alterations in D. melanogaster and, consequently, can be used to compare the susceptibility to genomic damage of different DNA repair mutants.     

Analysis of Vibrio mimicus clinical strains by arbitrarily primed polymerase chain reaction

A total of 51 Vibrio mimicus clinical strains from different geographic locations were examined by arbitrarily primed polymerase chain reaction (AP-PCR). The primer VMH-3 divided them into 28 groups, although 18 groups consisted of a single strain at present. All groups had a common 1.0-kb amplification fragment. Most of the groups consisted of strains from same region, although two exceptional groups showed a few amplification fragments including strains from different regions. AP-PCR groups were not consistently associated with serogroups. AP-PCR is thought to be a valuable and easy method for the epidemiological study of V. mimicus.     

Identification of a putative gamma-aminobutyric acid (GABA) receptor subunit rho2 cDNA and colocalization of the genes encoding rho2 (GABRR2) and rho1 (GABRR1) to human chromosome 6q14-q21 and mouse chromosome 4.

Screening of a genomic DNA library with a portion of the cDNA encoding the gamma-aminobutyric acid (GABA) receptor subunit rho1 identified two distinct clones. DNA sequencing revealed that one clone contained a single exon from the rho1 gene (GABBR1) while the second clone encompassed an exon with 96% identity to the rho1 gene. Screening of a human retina cDNA library with oligonucleotides specific for the exon in the second clone identified a 3-kb cDNA with an open reading frame of 1395 bp. The predicted amino acid sequence of this cDNA demonstrates 30 to 38% similarity to alpha, beta, gamma, and delta GABA receptor subunits and 74% similarity to the GABA rho1 subunit suggesting that the newly isolated cDNA encodes a new member of the rho subunit family, tentatively named GABA rho2. Polymerase chain reaction (PCR) amplification of rho1 and rho2 gene sequences from DNA of three somatic cell hybrid panels maps both genes to human chromosome 6, bands q14 to q21. Tight linkage was also demonstrated between restriction fragment length variants (RFLVs) from each rho gene and the Tsha locus on mouse chromosome 4, which is homologous to the CGA locus on human chromosome 6q12-q21. These two lines of evidence confirm that GABRR1 and newly identified GABRR2 map to the same region on human chromosome 6. This close physical association and high degree of sequence similarity raises the possibility that one rho gene arose from the other by duplication.     

Mitochondrial Genome Fingerprinting in Hybrid Rice and Its Associated Lines Using the Primed Polymerase Chain Reaction

A new economic and efficient DNA polymorphism assay was developed in 1990 that is based on the amplification by polymerase chain reaction (PCR) of random DNA segments using primers of arbitrary nucleotide sequence. Authors have now adapted this type of amplification to rice mitochondrial genome. Using 6 rice varieties in conjunction with 7 of 20–27 mer oligonucleotide primers, the AP-PCR products revealed that the amplified DNA bands fell into two categories, the evolutively conserved the cytoplasmic-specific. It is suggested that AP-PCR assay of mtDNA may help to classify or identify the cytoplasms in rice. By comparing "fingerprints" among the WA type cytoplasmic male sterility (CMS) rice, its Maintainer and Restorer lines, as well as its hybrid, one CMS cytoplasm-specific band (primer R2/630 bp) and one normal cytoplasm-specific segment (primer V5/707 bp) could be directly identified among the set of amplified DNA fragments. Further, some difference in the amplification patterns of mtDNA between CMS line and its hybrid, which infers that rearrangement of mitochondrial genome in hybrid rice probably happened.     

Chromosomal mapping of the human catechol-O-methyltransferase gene to 22q11.1----q11.2.

Catechol-O-methyltransferase (COMT; EC 2.1.1.6) is a physiologically important enzyme in the metabolism of catecholamine neurotransmitters and catechol drugs. Using primers derived from the known rat cDNA sequence for COMT, we have used the polymerase chain reaction to produce an amplified DNA fragment corresponding to the complete coding region of the rat gene. With this fragment as a probe, we have hybridized DNAs from two panels consisting of human/rodent and human/hamster somatic cell hybrids carrying various translocations and deletions to refine the chromosomal location of human COMT. Southern blot analysis indicates that the human COMT gene is localized to 22q11.1----q11.2, a region to which several anonymous DNA sequences, but until now, no structural genes, have been assigned.     

ARLTS1, MDM2 and RAD51 gene variations are associated with familial breast cancer

Genetic factors that contribute to the risk of breast cancer are largely not known and association studies have revealed several genes with low penetrance risk alleles for breast cancer. Analysis of these genes may provide important information on the risk factors affecting carcinogenesis. Variations in the ARLTS1, RAD51 and MDM2 genes have been associated with increased risk of different cancer types but for breast cancer the results are not consistent. In this study we investigated the role of the allelic variants in candidate genes acting in the tumor suppressor, DNA repair and p53 pathways as risk factors for familial breast cancer in 147 patients displaying characteristics of familial disease. Presence of the polymorphic variants were investigated by amplification of the corresponding regions and restriction fragment length polymorphism analysis. Genotype and allele frequencies in the patients were significantly different for all three variants. Our results indicate that the polymorphic variants might affect individual susceptibility towards breast cancer.     

Detection of amplified genomic sequences in human small-cell lung carcinoma cells by arbitrarily primed-PCR genomic fingerprinting

The arbitrarily primed-PCR (AP-PCR) genomic fingerprinting method was applied to evaluate its effectiveness in detecting and characterizing amplified DNA fragments in two small-cell lung carcinoma (SCLC) cell lines, NCI-H69 and NCI-H82. Of the 2428 DNA fragments detected by AP-PCR using 62 arbitrary primers, 2 (0.08%) DNA fragments were amplified in NCI-H69 and 6 (0.25%) DNA fragments were amplified in NCI-H82. Based on these results, we estimate the total size of the amplified genomic regions in these cell lines to be 3000 megabase pairs (Mb) × 0.0008 = 2.4 Mb in NCI-H69 and 3000 Mb × 0.0025 = 7.5 Mb in NCI-H82. The 2 amplified fragments in NCI-H69 were mapped to chromosome 2, and all 6 amplified fragments in NCI-H82 were mapped to chromosome 8. This strongly suggests that restricted chromosomal regions are specifically amplified in these SCLC cell lines. Since the N-myc gene at 2p24 is amplified in NCI-H69 and the c-myc gene at 8q24 is amplified in NCI-H82, it is possible that these DNA fragments are co-amplified with N-myc or c-myc in these cell lines. However, since the 2 amplified fragments in NCI-H69 were not amplified in 42 other human cancer cell lines including 11 cell lines carrying amplified N-myc genes, it is also possible that there are amplified regions on chromosome 2 other than the N-myc locus at 2p24 in NCI-H69. In contrast, all 6 amplified fragments in NCI-H82 were amplified in several other human cancer cell lines carrying amplified c-myc genes. This result further indicates that these fragments were derived from an amplification unit that includes the c-myc gene. Our results show the ability of the AP-PCR method to analyze the fraction of the genome with amplification in human cancer cells. Received: 10 April 1995 / Revised: 18 December 1995, 15 April 1996     

Alteration of a sequence with homology to human endogenous retrovirus (HERV-K) in primary human glioma: implications for viral repeat mediated rearrangement

We had earlier demonstrated that a comparison of DNA fingerprinting profiles of tumor and corresponding normal DNA from the same patient by random amplified polymorphic DNA (RAPD) analysis can readily demonstrate alterations in tumor DNA [Gene 206 (1998) 45 and J. Neuro Oncol. 48 (2000) 1]. These alterations could be used to identify changes in tumor DNA where the prior identity of the locus was not known. In this study, we report the identification, cloning and characterization of a RAPD amplified fragment which was lost in a glioma, a grade IV glioblastoma multiforme (GBM). Comparison of the RAPD profile of tumor and corresponding leucocyte DNA revealed several differences between the two. These included a band of 443 bases, which was demonstrated in the normal, but not in tumor DNA. On sequencing, this band was found to be homologous with a group of SINE sequences, which are probably derived from the human endogenous retrovirus-K (HERV-K). Homology search also reveals that HERV-K-derived sequences are interspersed, amongst others, in the tumor suppressor gene BRCA2 and the DNA repair gene XRCC1. Of particular interest is the inverted repeat pattern of HERV-derived sequences in the genes. While not demonstrating a cause effect relationship, this highlights the possible role of such virus-derived sequences in gene inactivation by recombination during tumorigenesis.     

DNA fingerprinting of human cell lines using PCR amplification of fragment length polymorphisms

Summary Methods for monitoring cell line identification and authentication include species-specific immunofluorescence, isoenzyme phenotyping, chromosome analysis, and DNA fingerprinting. Most previous studies of DNA fingerprinting of cell lines have used restriction fragment length polymorphism analysis. In this study, we examined the utility of an alternative and simpler method of cell line DNA fingerprinting—polymerase chain reaction (PCR) amplification of fragment length polymorphisms. Fourteen human cell lines previously found by other methods to be either related or disparate were subjected to DNA fingerprinting by PCR amplification of selected fragment length polymorphism loci. Cell identification patterns by this method were concordant with those obtained by isoenzyme phenotyping and restriction fragment length polymorphism-DNA fingerprinting, and were reproducible within and between assays on different DNA extracts of the same cell line. High precision was achieved with electrophoretic separation of amplified DNA products on high resolution agarose or polyacrylamide gels, and with fragment length polymorphism (FLP) loci-specific “allelic ladders” to identify individual FLP alleles. Determination of the composite fingerprint of a cell line at six appropriately chosen fragment length polymorphism loci should achieve a minimum discrimination power of 0.999. The ability of PCR-based fragment length polymorphism DNA fingerprinting to precisely and accurately identify the alleles of different human cell lines at multiple polymorphic fragment length polymorphism loci demonstrates the feasibility of developing a cell line DNA fingerprint reference database as a powerful additional tool for future cell line identification and authentication.     

A new semi-nested PCR protocol to amplify large 18S rRNA gene fragments for PCR-DGGE analysis of soil fungal communities

The analysis of soil fungal communities by molecular fingerprinting and subsequent identification of the underlying populations require the amplification of a phylogenetically informative gene fragment. In this study we tested the reliability and suitability of the previously published fungal primer combination (NS1/FR1-GC) that amplifies almost the entire 18S rRNA gene for the DGGE analysis of fungal communities in soil samples from 36 sites. This direct PCR system failed to amplify the fragment of interest from the total DNA extracted from most of the soils tested. Thus, we developed a new semi-nested PCR system based on the initial amplification of over 1,700 bp of the 18S rRNA gene with a new primer combination, followed by a subsequent amplification with NS1/FR1-GC. By means of the PCR approach developed in this study distinct 18S rRNA gene amplicons could be reproducibly generated for all soil samples. Amplification tests with 101 soil fungal isolates showed that with the new semi-nested system 18S rRNA gene fragments could be obtained from more fungi than with the direct approach. The subsequent DGGE separation of community amplicons resulted in a high resolution and revealed reproducible complex soil fungal communities specific for each site, despite a minor variability between replicates of the same sample. The semi-nested PCR system developed in this study, coupled with DGGE fingerprinting, offers a robust, reliable and sensitive tool for the analysis of soil fungal community structure.     

Amplification of CDK4, MDM2, SAS and GLI genes in leiomyosarcoma, alveolar and embryonal rhabdomyosarcoma

We evaluated amplification and overrepresentation of CDK4, MDM2, GLI and SAS genes of the 12q13-15 region, in a group of soft tissue sarcomas including leiomyosarcomas (LMS), alveolar rhabdomyosarcomas (ARMS) and embryonal (anaplastic and classic variants) rhabdomyosarcomas (ERMS), to ascertain genomic alterations and possible differences within histologic subtypes of rhabdomyosarcoma (RMS). Quantitative real-time PCR was performed on DNA samples from 29 LMS, 9 ARMS, 7 anaplastic ERMS and 6 classic ERMS. Alteration of one or more of the 12q13-15 genes was revealed in 13/29 LMS (45%) and 12/22 RMS (54%) including 5/9 ARMS (56%), 5/7 anaplastic ERMS (71%) and 2/6 classic ERMS (33%). The potential importance of overproduction of protein products in neoplastic development, led us also to study a possible high expression of cdk4, mdm2 and gli proteins in immunohistochemical staining experiments on paraffin-embedded tissue samples of the same cases. Among LMS and RMS most cases with CDK4, MDM2 and GLI gene alterations also showed a simultaneous high expression of the relative protein. In summary, these results indicate that amplification or overerepresentation of genes at 12q13-15 region involve both LMS and RMS. Moreover these genes alterations reveal predominantly in the alveolar and in the anaplastic variant of the embryonal subtype. These two seem to have a more similar behavior than anaplastic and classic embryonal that are classified in the same subtype.     

Segregation of the Huntington disease region of human chromosome 4 in a somatic cell hybrid

We have developed an X-irradiation:cell fusion procedure that segregates segments of human chromosomes lacking selectable markers and have used this approach to construct somatic cell hybrids retaining fragments of human chromosome 4 as the only human material. To identify hybrids retaining a small chromosomal fragment in the region of the Huntington disease (HD) gene, we used Southern blot analysis to screen 72 hybrid lines for the presence or absence of seven chromosome 4 single-copy loci. These data, combined with in situ hybridization experiments, identified three hybrids of interest. One of these cell lines, C25, stably retains a 10,000- to 20,000-kb fragment of distal 4p in the vicinity of the HD gene, translocated to a hamster chromosome. Field-inversion gel electrophoresis revealed no evidence of rearrangements in the human DNA present in C25. In combination with similar radiation hybrids, C25 is a valuable tool for isolating DNA probes near the HD gene.     

Complementation of repair gene mutations on the hemizygous chromosome 9 in CHO: a third repair gene on human chromosome 19

A human DNA repair gene, ERCC2 (Excision Repair Cross Complementing 2), was assigned to human chromosome 19 using hybrid clone panels in two different procedures. One set of cell hybrids was constructed by selecting for functional complementation of the DNA repair defect in mutant CHO UV5 after fusion with human lymphocytes. In the second analysis, DNAs from an independent hybrid panel were digested with restriction enzymes and analyzed by Southern blot hybridization using DNA probes for the three DNA repair genes that are located on human chromosome 19: ERCC1, ERCC2, and X-Ray Repair Cross Complementing 1 (XRCC1). The results from hybrids retaining different portions of this chromosome showed that ERCC2 is distal to XRCC1 and in the same region of the chromosome 19 long arm (q13.2-q13.3) as ERCC1, but on different MluI macrorestriction fragments. Similar experiments using a hybrid clone panel containing segregating Chinese hamster chromosomes revealed the hamster homologs of the three repair genes to be part of a highly conserved linkage group on Chinese hamster chromosome number 9. The known hemizygosity of hamster chromosome 9 in CHO cells can account for the high frequency at which genetically recessive mutations are recovered in these three genes in CHO cells. Thus, the conservation of linkage of the repair genes explains the seemingly disproportionate number of repair genes identified on human chromosome 19.     

In-solution hybrid capture of bisulfite-converted DNA for targeted bisulfite sequencing of 174 ADME genes

DNA methylation is one of the most important epigenetic alterations involved in the control of gene expression. Bisulfite sequencing of genomic DNA is currently the only method to study DNA methylation patterns at single-nucleotide resolution. Hence, next-generation sequencing of bisulfite-converted DNA is the method of choice to investigate DNA methylation profiles at the genome-wide scale. Nevertheless, whole genome sequencing for analysis of human methylomes is expensive, and a method for targeted gene analysis would provide a good alternative in many cases where the primary interest is restricted to a set of genes.Here, we report the successful use of a custom Agilent SureSelect Target Enrichment system for the hybrid capture of bisulfite-converted DNA. We prepared bisulfite-converted next-generation sequencing libraries, which are enriched for the coding and regulatory regions of 174 ADME genes (i.e. genes involved in the metabolism and distribution of drugs). Sequencing of these libraries on Illumina’s HiSeq2000 revealed that the method allows a reliable quantification of methylation levels of CpG sites in the selected genes, and validation of the method using pyrosequencing and the Illumina 450K methylation BeadChips revealed good concordance.     

A somatic cell hybrid panel and DNA probes for physical mapping of human chromosome 7p.

To identify by reverse genetics genes on the short arm of human chromosome 7 expected to be involved in the regulation of human craniofacial and limb development, we have set up a human mouse somatic cell hybrid panel that divides 7p into 9 fragments. The breakpoints are defined by deletions or translocations involving one chromosome 7 in the cells of the human cell fusion partners. Particularly densely covered with these cytogenetic anchor points is the proximal area of 7p within and around 7p13. The number of cytogenetic mapping points within proximal 7p could be increased by four, using two diploid human cell lines with small interstitial deletions in this region for dosage studies. We used Southern blots of this panel to assign to 7q or subregions of 7p more than 300 arbitrary DNA probes or genes that provide reference points for physical mapping of 7p. Three reciprocal translocations with one of the breakpoints in 7p13 mark the location of a gene involved in Greig cephalopolysyndactyly syndrome. To define an area in which we could identify candidates for this developmental gene, we established a macrorestriction map using probes flanking the putative gene region. The Greig translocations were found to be located within a 630-kb NotI restriction fragment.     

Chromosomal assignment of 38 human brain expressed sequence tags (ESTs) by analyzing fluorescently labeled PCR products from hybrid cell panels.

We have localized 38 human brain cDNA sequences to individual human chromosomes. PCR primers were designed from expressed sequence tags and tested for specific amplification from human genomic DNA. The sizes of amplification products from DNA of somatic cell hybrid mapping panels were determined electrophoretically using an automated fluorescence detection system. Chromosomal assignments were made by discordancy analysis.